How the Left Are Enabling ISIS & the Far-Right

Socialists and consistent democrats who do not condemn political Islam, its methods, its objectives and the casual mass slaughter which it employs have lost their political bearings. Completely. Those who deduce from hostility to the USA support for, or a refusal to condemn, Islamic terrorism are so warped by negativism toward capitalism and imperialism that they forget, if they ever knew, what socialists and consistent democrats are, positively, for. Political Islam is the enemy of everything socialists stand for, and its bloody tactic of indiscriminate mass murder mirrors what it is. In the first place, it is the enemy of the working class and of all the women of countries where Islam predominates.

Sean Matgamna, Worker’s Liberty



Prologue: A Note on the French Elections

The regime of Francois Hollande was marked by a series of measures that stripped away workers’ rights. The incumbent Emmanuel Macron was at the heart of this, being Minister of Economy in Hollande’s cabinet: indeed, the ‘Macron Law’ not only simplifies labour tribunals such that they favour employers, but allows those employers to offer severance packages which don’t actually reflect the financial strength of the parent company. As President, Macron now plans to sack 120,000 civil servants and to cut public spending by €60 billion. He also plans to cut corporation tax from 33 percent to 25 percent, and is a passionate advocate for trade deals such as TTIP and CETA, which enable corporations to sue the State for profits lost owing to industrial action.

With all this in mind, there is surely a potential for mass protests in the coming months by both public and private sector workers. With the centre-right and the centre-left discredited, the choice before the French people may well be between Marine Le Pen and Jean-Luc Melenchon. What might swing things in Le Pen’s favour is the likelihood of further terror attacks: for Macron has pledged not only to keep extremist mosques open, but to maintain trade with Saudi Arabia, the chief exporter of Islamic terror. As things stand, France is the chief exporter of weapons to Saudi Arabia, to the tune of €2 billion a year – six times that of the United Kingdom.

Le Pen talks tough about Islamism and Western values, even if she is no follower of the Enlightenment: the record of the Front Nationale proves this, from their holocaust denial to their rampant homophobia. Her rhetoric resonates with people, however, because no one else is taking up that mantle. For the Left to win, therefore, Melenchon needs to identify Islamism as an evil in and of itself, and to challenge it as an enemy of social progress – just as he does with neoliberalism. If he does that, then to defeat Le Pen, he just needs to keep on doing what he’s doing. If he doesn’t, then fascism could well be on its way back, barely seventy years after Nazi Germany.



Article

It is a disturbing fact that, for many on the Left, the power of dogma is such that the likes of ISIS escape sincere criticism. Being Marxists, these people cannot accept that religious belief ever trumps economics; thus, ISIS are understood not as religious fanatics with a divine mandate, but as “misguided anti-capitalists” fighting to end the rule of the 1 percent. When confronted with the sectarian atrocities of these groups, to avoid having to question their faith, the response is simply to point out the crimes of Western governments, with the charge of “Islamophobia” being kept at arm’s length.

To describe these people, Maajid Nawaz of the Quilliam Foundation has coined the phrase the ‘Regressive Left’. In his words, these are Leftists who “abuse terms such as racism, bigotry and Islamophobia to shutdown debate”. In an interview with News7, Nawaz explains how the Regressive Left is enabling Wahhabi-Salafi extremists to continue running the show: for when we deny that terrorism has anything to do with Islam, we deprive liberal Muslims and ex-Muslims of the authority they need to modernise the faith:

It is self-evident that ISIS have got something to do with Islam. When ISIS throws gays off the top of buildings, they are using scripture. In fact, there are traditions ascribed to the Prophet where it says that is exactly the punishment that should be given to gays. When they are stoning women to death, it is in scripture. When they are chopping off hands, I can read you the Arabic in the Qur’an which tells them to do that…

If you don’t recognise that Islam is being used by megalomaniac, totalitarian theocrats for their own project, what you are doing is depriving Muslim reform voices of the authority they need to reclaim their faith from those who are hijacking it. Muslims are complaining that their faith is being hijacked; well, if you are saying your faith is being hijacked, clearly it is something to do with Islam then. It is a disingenuous stance to take otherwise and it hinders the voice of moderate Muslims looking towards reform.

Nawaz also exposes the hypocrisy of those Muslims who claim that they shouldn’t have to condemn ISIS, and yet expect Westerners to condemn anti-Muslim bigotry outright. The belief here, of course, is that non-Muslims are inherently prone to evil, whereas Muslims are inclined towards righteousness:

My fellow Muslims are slightly defensive about discussing extremism, and yet we would find it infuriating if a white man, in a discussion about racism, said “Why should I have to condemn racism?” Some Muslims will say “Why should I have to condemn extremism?”, but will expect solidarity from mainstream society in the face of anti-Muslim bigotry and racism. That is a double standard.

As Sean Matgamna has put it, political Islam is the enemy of everything that socialists stand for – or at least, socialists who haven’t totally lost their minds. Indeed, if ISIS were to assume the position of government in Ireland, socialists would be the first ones put to death for their atheism, their secularism, and for advocating gay marriage. They don’t seem to understand that, when it comes to politics, Islamic fundamentalists have a lot more in common with the far-Right than themselves. They are quite ready to label Donald Trump as a fascist (or equivalent), but would never dare use this term in reference to Ali Selim or Hussein Halawa, both of whom are quite open about their desire for shari’a law in Ireland. To quote Christopher Hitchens on the striking similarities between fascism and Wahhabi-Salafism:

Does Bin Ladenism or Salafism or whatever we agree to call it have anything in common with fascism? I think so, yes. The most obvious points of comparison would be these: Both movements are based on a cult of murderous violence that exalts death and destruction and despises the life of the mind… Both are hostile to modernity (except when it comes to the pursuit of weapons), and both are bitterly nostalgic for past empires and lost glories. Both are obsessed with real and imagined “humiliations” and thirsty for revenge. Both are chronically infected with the toxin of anti-Jewish paranoia (interestingly, also, with its milder cousin, anti-Freemason paranoia). Both are inclined to leader worship and to the exclusive stress on the power of one great book. Both have a strong commitment to sexual repression – especially to the repression of any sexual “deviance” – and to its counterparts the subordination of the female and contempt for the feminine. Both despise art and literature as symptoms of degeneracy and decadence; both burn books and destroy museums and treasures.

When the Left refuses to recognise these simple truths, this lends credibility to far-Right groups, who use the immigration issue as a cover for racist bigotry. When the Left pretends that terrorism has nothing to do with Islam, which is so obviously false, the far-Right are able to use our silence to rally people under the banner of “saving Western civilisation”, which, for the most part, is just a cover for keeping Europe white. This is the sworn mission of those who believe culture to be dependent on race, e.g. PEGIDA and Sons of Odin, who tend not to be well-versed in Enlightenment philosophy, to say the least. In reality, values such as humanism and rationalism depend on brains for their existence, not blood: I would much sooner befriend an Inuit who has learned the art of survival, for example, than a Confederate hick who takes pride in his scientific illiteracy.

It is imperative that individuals are held responsible for racist crimes: for once that beast is let loose, violence and oppression always increase exponentially. Just as imperative, however, is to recognise that if the Left stood firmly against political Islam, then fewer people would be attracted to the far-Right. If the Labour Party in Britain took a principled stance against open-door Muslim immigration, if they denounced the operation of Wahhabi-Salafi mosques and explicitly condemned shari’a law, then UKIP would barely have a leg to stand on. The Irish Left ought to take heed of this simple truth: for if they don’t, then given the growing presence of ISIS in our mosques, schools and campuses, it is only a matter of time before the far-Right coalesces in this country. To quote Dr. Ali al-Saleh of the Shi’a Mosque in Milltown, who are prevented by threats from the Clonskeagh Mosque (Salafist) from having a sign up identifying it as such:

Members of ISIS live here, they are active at the level of small circles, giving lectures, talking to the youth. This is a problem. We’ve said that from the beginning, now we have it. We didn’t tackle it from the beginning. It is our duty as Imams to talk openly against those things… I’m very concerned that some Muslim organisations in colleges are dominated by radical thinkers who are brainwashing the minds of students. Where you see them holding lectures on Palestine, Gaza, Libya, you have to ask what their motivation is. Where we see the niqab and the burqa, that is another symbol of extremism and the repression of women. We want our students to concentrate on their studies and learn from the West about how to bring democracy to their countries, not to come here and become radicalised.



Postscript: Anti-Putin Protests & the Barbarity of ISIS

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Tallaght Stadium, October 2016: members of the Irish Syria Solidarity Campaign protest against Russian airstrikes. Of course, these same people are nowhere to be seen at anti-ISIS protests, despite the latter’s campaign of genocide against Christians, Jews, Yazidis, Shi’a Muslims and Sunni moderates in Iraq and Syria. Their silence could not be more deafening, dear reader. Have a read of the following excerpts from a 2014 Amnesty International report, detailing the true horror of ISIS:

In the afternoon of August 3rd, scores of men and boys were summarily killed on the edge of the village of Qiniyeh, south-east of the town of Sinjar. Amnesty International spoke to several survivors and witnesses of the massacre, all of whom gave very similar accounts. They were interviewed separately and in some cases did not know each other. According to their statements, a large group of some 300 or more Yazidis, most of them from the nearby village of Tal Qasab and many from the same extended family and tribe, became trapped in Qiniyeh as they were making their way to Mount Sinjar. Most of them were women and children, but there were also scores of men, many of whom had earlier engaged in armed clashes with IS fighters, in an attempt to prevent them from storming their villages.

Once they realised they had no hope of halting the IS advance, they fled north towards Mount Sinjar, getting stuck in Qiniyeh. There, IS fighters caught up with them, separated the women and children from the men and boys and took a group of up to 85-90 men and boys – including boys as young as 12 – and shot most of them dead. The massacre seems to have been carried out to punish those who had, or were suspected of having, tried to repel the IS attack on their villages, and/or to dissuade others from putting up any resistance to IS advances. A few people survived the massacre, and some others managed to escape in the melee as they were being marched to the killing site. Amnesty International spoke to some of the survivors. One villager, Mohsen Elias, told Amnesty International:

“After the Peshmerga who used to protect our villages fled in the night between the 2nd and 3rd of August, me and many other men from the village took our weapons – most of us had Kalashnikovs, for the protection of our families – and clashed with IS militants. At about 7 or 8am we ran out of ammunition and ran away toward the mountain. We stopped in the village of Qiniyeh, near the foot of the mountain. We were about 90 men and youths and with us were more than 100 women and children from our families.”

“At about 1pm or so, IS militants came and spoke to us and said that they were only looking for Peshmerga. They asked if we had weapons and said they would kill anyone found to have weapons. We had hidden our weapons and said we had none. They said we could go home soon and left. After half an hour, some 20 IS vehicles came and surrounded us. My relative Nasser Elias tried to run away and they shot him dead.”

“They split us into two groups, men and boys of 12 and older in one group and women and younger children in another group. They started to load the women and children in the vehicles and made us walk to the nearby wadi. The youngest of the group was my brother Nusrat, 12 years old. We were made to squat by the edge of the wadi, which was deep. They told us to convert to Islam and we refused.”

“One grabbed me by my shirt from behind and pulled me up and tried to shoot me, but his weapon did not fire. My brother Nusrat was scared and was crying. They opened fire from behind us. I fell into the wadi and was not injured. My brother Nusrat was right next to me and was killed. My father, Elias, and my four brothers, Faysal, Ma’amun, Sa’id and Sofian, were all killed. Most of the other men and boys were also killed, including more than 43 of my relatives. After the IS men left, I waited and then ran away to the mountain. I only know four others who survived: my neighbour Fawas, Khalaf Mirze and his son ‘Ayad, and another man called Ziad. I don’t know if any others survived.”

On Islamic Imperialism, Terror & the Need for Ijtihad

I shall cross this sea to their islands to pursue them until there remains no one on the face of the earth who does not acknowledge Allah.

Saladin, January 1189

I was ordered to fight the people until they say there is no god but Allah, and his prophet Muhammad.

Osama bin Laden, November 2001



Contrary to what many on the Left seem to think, pointing out the crimes of Christianity is not a sufficient response to the scourge of Islamic terrorism. Whereas Christian belligerence has receded with the demise of the old European empires, its Islamic counterpart has endured to this day, a century after the fall of the Ottoman Empire. This is because the religion is inherently imperial, whereas Christianity only became so when it was adopted by the Roman Empire for convenience. This imperial disposition is cemented by the doors of inquiry (ijtihad) being sealed shut centuries ago, during the period of the Abbasid Caliphate. Until the Muslims of the world collectively decide to reopen those doors, their situation will never truly improve. To this end, Western people should be assisting Muslims in evaluating their religion, not cowering in fear of it. In the words of Muhammad Ibrahim Jannati, a Shi’a Muslim scholar:

The cause of the present inadequacies of Sunni fiqh in most of the branches of law, as well as the issuance of incorrect fatwas, is the Sunni belief in the end of ijtihad. The committed and aware scholars among the Ahl al-Sunnah [Sunni Muslims] should endeavour to reopen the gates of ijtihad and bring Islam out of its current state of isolation and decadence, so that a spirit of vitality and dynamism is infused into Muslim communities. This is because so long as the taqlid of the four Imams is considered binding, and new research, study and expression of views is regarded as impermissible, there appears to be little hope of any effective change.

In this article, we will touch on how imperialism, far from being alien to Islam, is endemic to the emergence and spread of the faith. We will argue that terrorism, being a tactic employed by Muslims to achieve the Islamic State, can only be overcome by removing the desire for theocracy, i.e. by undermining the certainty of their convictions, thus necessitating the rebirth of ijtihad. Indeed, it is the absence of doubt that makes the likes of ISIS so dangerous: for if suicide bombers weren’t totally convinced of the life to come, they would hardly go through with it. As the philosopher Will Durant once said, intolerance is the natural concomitant of strong faith; tolerance grows only when faith loses certainty. To quote Sam Harris on the power of belief:

Believe that you are the member of a chosen people, awash in the salacious exports of an evil culture that is turning your children away from God, believe that you will be rewarded with an eternity of unimaginable delights by dealing death to infidels – and flying a plane into a building is scarcely more than a matter of being asked to do it.


Part I

Islamic Imperialism: A Millennial Tradition

Open any modern introductory book on Islam, dear reader, and chances are that it begins by singing the praises of a people who conquered, in an incredibly short period, half the civilised world; of a people who established an empire that stretched from the banks of the Indus River to the shores of the Atlantic Ocean. The book will recount, in positively glowing terms, a time when Muslims ruled over a vast population of diverse peoples and cultures. One can hardly imagine a contemporary British historian getting away with making similar eulogies of the British Empire. While European colonialism and imperialism are blamed for every ill on earth, Arab imperialism is held up as something of which Muslims can be proud, an achievement to be lauded and admired.

This tendency strongly informs the debate about Islam and terrorism today. Indeed, while the 9/11 attacks have inspired various interpretations of their root causes, ultimately, the two prevailing schools of thought assign the blame to the West. According to the first school, the attacks were but the latest saga in the ancient clash between the worlds of Islam and Christendom; a violent backlash by a deeply frustrated civilisation reluctant to accept its long-standing decline. The historian Bernard Lewis writes as follows:

For many centuries Islam was the greatest civilisation on earth – the richest, the most powerful, the most creative in every significant field of human endeavour. And then everything changed, and Muslims, instead of invading and dominating Christendom, were invaded and dominated by Christian powers. The resulting frustration and anger at what seemed to them a reversal of both natural and divine law have been growing for centuries, and have reached a climax in our own times.1

Not so, argues a vast cohort of academics, journalists, writers, and retired diplomats. The attacks were a misguided response to America’s self-serving foreign policy by a fringe extremist group, whose violent interpretation of Islam has little to do with the actual faith. “Muslims have never dreamed of world conquest”, Karen Armstrong argues:

They had no designs on Europe, for example, even though Europeans imagined that they did. Once Muslim rule had been established in Spain, it was recognised that the empire could not expand indefinitely.2

Neither of these interpretations is particularly novel. Both echo the traditional wisdom in the field of Middle Eastern studies, which views the region’s history as an offshoot of global power politics. Both essentially agree that Western imperialism bears the main responsibility for the cultural and religious malaise plaguing the Middle East to date.

In reality, this malaise is merely the culmination of long-existing indigenous trends, passions and behavioural patterns – first and foremost being the region’s imperial tradition, expressed most emphatically by the Islamic conquests. External influences, however potent, have played only a secondary role, constituting neither the primary force behind the Middle East’s political development nor the main cause of its volatility.

Contrary to the conventional wisdom, it is the Middle East where the institution of empire not only originated (e.g. Egypt, Assyria, Babylon, Iran), but where its spirit has also outlived its European counterpart. At the time of the rise of Islam in the early seventh century, the Middle East was divided between the two great rival empires of Byzantium, the successor to the Roman Empire with its capital in Constantinople, and Iran, ruled since the third century by the Sasanid dynasty with its capital in Ctesiphon, where Baghdad stands today.

Though the Arabian Peninsula, birthplace of Islam, was on the fringes of this bipolar system, it was deeply implicated in its activities. The Iranians had colonies throughout eastern and southern Arabia, in Najd and in Yemen, and their influence extended all the way to the Hijaz, the north-western part of the peninsula. Byzantium’s power was felt throughout western Arabia from the Syrian Desert, where it had client kingdoms, to Yemen, where its Ethiopian allies had ruled until they were expelled by the Iranians.3

This ancient tradition of imperialism, entrenched as it was in master and subject alike, meant that for Islam to make its mark in the Middle East, it was destined to follow a very similar path. Knowing this, to obscure the imperialist motive behind the early Islamic conquests, apologists for Islam insist that any examination of the rise of the faith and its spectacular success must ignore the impact of Byzantium and Iran on Arabia. One possible way to do so would be to view nascent Islam as

a nativist movement, or in other words as a primitive reaction to alien domination of the same types as those which the Arab conquerors were themselves to provoke in North Africa and Iran, and which European colonists were later to provoke throughout the Third World…the object of the movement being the expulsion of the foreigners in question.4

In reality, of course, there is a massive difference between conquest and resisting occupation. Expelling occupiers from one’s homeland is an act of self-liberation. Conquering foreign lands and subjugating their populations is pure imperialism. Neither North African Berbers fighting their Islamic conquerors nor Third World movements resisting European colonialism aspired to conquer the homeland of their imperial masters. Yet this is precisely what Muhammad asked of his followers once he had fled from his hometown of Mecca (in 622 A.D.) to the town of Medina to become a political and military leader: not to rid themselves of foreign occupation, but to strive for a new universal order in which humanity would either embrace Islam or live under its domination. As he told his followers in his farewell address:

I was ordered to fight all men until they say, “There is no god but Allah.”5

This appealed to the warlike spirit of the Arabian nomads. By the time of Islam’s emergence, with trade routes being increasingly harassed by marauders, violence had become endemic to Arabian society. This was vividly illustrated by the numerous fratricidal clashes known as Ayyam al-Arab (the Days of the Arabians), arising from disputes over such material issues as cattle, pasture-lands or springs, and glorified by contemporary poetry as an ideal of heroism and manhood. As one pre-Islamic poet boasted:

We slew in requital for our slain an equal number and [carried away] an unaccountable number of fettered prisoners. The days have thus raised us to be foremost with our battles in warfare; men find in us nothing to point their finger of scorn.6

No single activity of pre-Islamic society epitomised this warlike ethos more succinctly than the razzia (raid), a marauding expedition aimed at seizing camels, horses, livestock, or, less frequently, women from a hostile tribe. For most Bedouins, the razzia was not merely a means of subsistence, but rather an exciting social endeavour that helped reaffirm tribal solidarity and win invaluable military experience. In fact, the razzia was so prolific that scholars such as Montgomery Watt have described it as the “national sport” of the nomadic Arabians.7

Indeed, Muhammad initially devised the concept of jihad not as an internal struggle for self-improvement, but as a means to entice his local followers to raid Meccan caravans, thus transforming a common tribal practice into a supreme religious duty. He developed and amplified this concept with the spread of Islam until it became a rallying call for world domination, and he established the community of believers (umma) as the political framework for the practice of the religion in all territories it conquered.

By tapping into the Middle East’s millenarian legacy, Muhammad ensured its perpetration for over 1,400 years to come. From the first Arab-Islamic empire of the mid-seventh century to the Abbasid Caliphate, from the Ottoman Empire to Islamic State (ISIS), the story of Islam has been marked by the rise and fall of universal empires. Overextension leads to inadequate means of communication and control; thus, the determined pursuit of absolutism is matched by the equally formidable forces of fragmentation and degeneration. This has happened with Islamic State, whose impending collapse reflects an inability to maintain a foothold in both Iraq and Syria.

In the long history of Islamic imperialism, the force of arms has proven to be a recurring theme, making violence a key element of Islamic political culture. No sooner had Muhammad died than his disputed successor, Abu Bakr, had to suppress a widespread revolt among the Arabian tribes. Twenty-three years later, the Caliph Uthman ibn Affan was murdered by disgruntled rebels; his successor, Ali ibn Abi Talib, was confronted for most of his reign with armed insurrections – most notably by the governor of Syria, Mu’awiyah ibn Abu Sufyan, who went on to establish the Umayyad dynasty after Ali’s assassination.

Mu’awiyah’s successors clung to power mainly by using physical force – including his son, Yazid ibn Mu’awiyah, who was killed in a populist uprising after ordering an army battalion to slaughter Hussain ibn Ali, the grandson of the Prophet, for highlighting the illegitimacy of his rule. This violent tendency was equally becoming of the Abbasids during the long centuries of their reign, and is what ultimately led the Ottoman Empire to enter World War I on the losing side. Needless to say, the imperialist dream of the Caliphs has survived the Ottoman era to haunt Islamic and Middle Eastern politics to this day.


It is true, of course, that the pattern of historical development we have described is not uniquely Middle Eastern or Islamic. Other parts of the world, Europe in particular, have had their share of imperial powers and expansion. The worlds of Christianity and Islam, however, have developed differently in one fundamental respect. Put simply, the Christian faith was not imperial in its origin: rather, it became so when it was embraced by the Byzantine emperors to buttress their imperial claims, three centuries after its foundation. Its universalism was originally conceived in purely spiritual terms, making a clear distinction between God and Caesar. To quote from Matthew 22 (KJV):

And he [Christ] saith unto them, “Whose is this image and superscription?”

They say unto him, “Caesar’s.” Then saith he unto them, “Render therefore unto Caesar the things which are Caesar’s; and unto God, the things which are God’s.”

When they had heard these words, they marvelled, and left him, and went their way.

Contrast this with Islam, whose birth was inextricably linked with the creation of an empire, and whose universalism was inherently imperialist. It did not distinguish between temporal and religious powers, which were combined in the person of Muhammad, who derived his authority directly from God and who acted simultaneously as both head of church and state. This allowed the Prophet to cloak his political ambitions with a religious aura, and to channel Islam’s energies into “its instrument of aggressive expansion, there [being] no internal organism of equal force to counterbalance it.”8

Whereas Jesus spoke of the Kingdom of God, Muhammad used God’s name to build an earthly kingdom. He spent the last ten years of his life fighting to unify Arabia under his reign. Had it not been for his sudden death in 632 A.D., he would most probably have expanded his rule well beyond the Arabian Peninsula. Even so, within a decade of Muhammad’s death, a vast empire had come into being under the banner of Islam, stretching from Iran to Egypt and from Yemen to northern Syria.

This fundamental difference between the two faiths means that, whereas Christianity had lost its imperial ambitions by the mid-twentieth century, Islam has retained them to this very day, long after the fall of the Ottoman Empire and the abolition of the Caliphate in the wake of World War I. (This situation is compounded by the loss of ijtihad, which we will come to in part two.)


The eminent Dutch historian Johannes Kramers once commented that in medieval Islam, there were never real states, but only empires more or less extensive, and that the only political unity was the ideological concept of the House of Islam (Dar al-Islam), the common “homeland” of all Muslims.9 This observation can also be applied to the post-World War I era, where the two contending doctrines of Islamism and pan-Arabism have sought to fill the vacuum left by the collapse of the Ottoman Empire. Of these two, Islamism has been the greater success: for while pan-Arabists content themselves with a more modest empire comprising the Middle East, Islamism views this development as a mere prelude to the creation of a Muslim-dominated world order – a far more comforting belief for Muslims who resent the success of an increasingly godless Western civilisation.

The European empires of old were, by and large, overseas entities that drew a clear dividing line between master and subject.10 With their demise, therefore, formerly subject peoples were able to develop their own distinct brands of state nationalism, from Africa to South America. This is in stark contrast to the Islamic empires, which conquered other Arabic-speaking nations and which employed religion to blur the distinction between the ruling and ruled classes. Consequently, since the fall of the Ottoman Empire, Middle Eastern people have preferred to see themselves as members of a universal “Islamic umma” rather than patriots of their own specific nation-states. (Egypt is only a partial exception to this, given the election of the Muslim Brotherhood in 2012. As for Libya, ever since the fall of Gaddafi, Islamist groups have essentially been running the show.)

When we examine the diverse, fragmented tribes of the Arabic-speaking world, it is clear that these nations are more disposed to local patriotism than to a unified regional order. Alas, rather than allowing this disposition to run its natural course and develop into modern-day state nationalism, Islamist ideologues have systematically convinced their peoples to think of their respective states as a temporary aberration, to be rectified with the reestablishment of the Caliphate. The result has been a violent streak that has plagued the Middle East well into the twenty-first century, ensuring that ISIS meets with no substantial resistance by the mainstream. This brings us nicely to part two.



Part II

Terrorism & the Need for Ijtihad

Strictly speaking, ijtihad is the process of making a legal decision on matters which are not explicitly mentioned in the Qur’an or the hadith. More broadly, it refers to the use of reason in appraising the hadith literature as a sound basis for interpreting the Qur’an. When we discuss the loss of ijtihad, it is mainly the latter definition we are referring to. The aforementioned Shi’a scholar Muhammad Ibrahim Jannati describes ijtihad as follows:

In Islamic fiqh, ijtihad has had the meaning of a free and independent effort of the mujtahidun [scholars], undertaken for the purpose of advancement and expansion of the Islamic sciences. This development was not possible except through freedom of scholarly research, free expression of different views, and clashes between views of the mujtahidun…

The present deficiencies in the world of Islam, the issuing of baseless and irrational fatwas, the improper attitude towards new ideas – all these are consequences of the closing of the gates of ijtihad by the Ahl al-Sunnah [Sunni Muslims]. It has been instrumental in allowing dubious hands to instil unhealthy ideas into people’s minds and to insinuate the feeling that Islamic fiqh cannot fulfil the demands of modern civilisation…

The most outstanding achievement of Shi’a fiqh has been to keep open the gates of ijtihad throughout the course of history. Its superiority over other schools of fiqh, whose doors were closed after the death of their founders (namely, Abu Hanifah al-Nu’man ibn Thabit, the founder of the Hanafi school; Malik ibn Anas al-Asbahi, the founder of the Maliki school; Muhammad ibn Idris al-Shafi’i, the founder of the Shafi’i school; and Ahmad ibn Hanbal, the founder of the Hanbali school), lies here.

The loss of ijtihad during the era of the Abbasid Caliphate (750 – 1258) is what prevents Islam from discarding its imperial goals. The uncritical acceptance of the hadith literature by Sunni Muslims, in which countless exhortations to martyrdom and violence abound, makes the faith a breeding ground for terrorism: for the hadith is the primary basis for interpreting the Qur’an. Indeed, the Qur’anic commentaries of the medieval warmonger Ibn Taymiyyah (d. 1328) lie at the heart of modern Islamic terror. These commentaries, modernised by the Najdi preacher Muhammad ibn Abd al-Wahhab, are the foundation of Wahhabi-Salafism – the common ideology of ISIS, Al-Qaeda, the Taliban, Boko Haram, Al-Shabaab, and countless other militant groups hell-bent on spreading Islam by the sword.

Though the likes of Tariq Ramadan hate to admit it, the loss of ijtihad means that Wahhabi-Salafism is no less legitimate than any other interpretation of Sunni Islam. The lack of critical inquiry into the hadith literature precludes any real understanding of how Muhammad understood the Qur’an. This is crucial, for although Muhammad was an explicit imperialist, the lack of an authoritative exegesis (tafsir) means that more militant interpretations are given legitimacy, such as that of Ibn Taymiyyah. This academic free-for-all is the reason you don’t see Muslims protesting en masse against terror attacks, from Nice to St. Petersburg: for they cannot denounce the likes of ISIS without denouncing Ibn Taymiyyah, renowned to this day as “the great Islamic revivalist”.

Yes, until the Sunni Muslims of the world decide on the exclusive legitimacy of one tafsir, the commentaries of Ibn Taymiyyah will continue to hold water, and so the plague of Islamic terror will continue unabated. Arriving at that authoritative text necessitates a critical analysis of the hadith literature, which tafsirs rely upon for explaining each verse. By filtering out weak or unsupported hadith, we help to curb political explanations for contentious verses, thereby limiting the possibilities for terrorism. This is why you do not come across Shi’a Muslims – who keep ijtihad alive by comparing the hadith with the traditions of Muhammad’s family (Ahlul-Bayt) – using the following verse as justification for suicide bombings:

Let those fight in the cause of God who barter the life of this world for that which is to come; for whoever fights on God’s path, whether he is killed or triumphs, We will give him a handsome reward. (4: 74)

In contrast, Sunni Muslims regard their main sources of hadith as indisputably authentic. This leads to all manner of dubious interpretations, for a substantial number of hadith were concocted by the Caliphs for political gain (see the notorious forgerer Abu Hurayrah), with countless more being reported inaccurately or only in part. Since they are all considered reliable, there is more than enough material for fascists like Ibn Taymiyyah to work with. Here is what the man thought of suicide as a means of warfare:

Muslim has narrated in his Sahih the story of the people of the trenches, in which the boy ordered his own killing for the benefit of the religion, and hence the four imams have allowed a Muslim to immerse himself in the enemy ranks, even if he is reasonably certain that they will kill him, provided there is benefit in that for the Muslims.11

This ruling has inspired countless suicide bombings by ISIS and other Wahhabi-Salafi groups. They have the full backing of Sheikh Gibril Haddad of Sunni Path, who has stated that “there is no legal difference between the direct hand of the self-detonating suicide fighter and the proxy hand of the outnumbered fighter entering the fray alone.” Ibn Taymiyyah was no less fanatical towards Muslims who didn’t fully embrace his militant approach, as this ruling demonstrates:

Every party that resists the manifest and definite laws of Islam from these people [Tatars] or others, then it is obligatory to fight them until they comply with its laws even if they pronounce the shahada [declaration of faith] and follow some of its laws, just as Abu Bakr and the Sahabah [companions] fought those who resisted the Zakah [donation]… Any party that resists some of the obligatory Prayers, Fasting, Hajj, or resists abiding by the prohibition of spilling blood, looting wealth, alcohol, gambling, incest, or resists adherence to Jihad against the kuffar [non-believers] or the enforcement of Jizyah [protection tax] upon the Ahlul-Kitab [Jews and Christians], or abiding by anything else of the obligations and prohibitions of the Deen [faith], those rulings which no one has an excuse for being ignorant of or abandoning and which the individual commits kufr [disbelief] by denying, then the resistant party is fought over these rulings even if it acknowledges them. This is something of which I know no difference between the Scholars.12

In other words, if someone becomes a Muslim and acts like a Muslim, but there are some issues on which they disagree with Ibn Taymiyyah, then that person should be fought until he either submits or is killed for his disbelief. This is the justification for the deaths of so many Shi’a Muslims and moderate Sunnis (including Kurds) by the hands of ISIS: that they are not “real” Muslims because they don’t share their particular interpretation of Islam. This extreme intolerance applies to non-Muslims as well – the Druze in particular, whom Ibn Taymiyyah says should be killed “wherever they are found”:

Pic 1

The brutal persecution of Yazidis, Jews and Christians by ISIS directly follows the dehumanising logic of Ibn Taymiyyah. The mistreatment of non-Muslims living under Islamic rule is well-documented, and its pattern is always the same: after conquest, the men taken with arms are slain or sold into slavery, with their wives and children reduced to servitude. As for the non-combatants among the vanquished, if they are not massacred outright, it is only to give them respite until they are wise enough to embrace Islam. To quote Sir Jadunath Sarkar in this regard:

The conversion of the entire population to Islam and the extinction of every form of dissent is the ideal of the Muslim State. If any infidel is suffered to exist in the community, it is as a necessary evil, and for a transitional period only… A non-Muslim therefore cannot be a citizen of the State; he is a member of a depressed class; his status is a form of slavery. He lives under a contract (zimma, or dhimma) with the State: for the life and property grudgingly spared to him by the commander of the faithful, he must undergo political and social disabilities, and pay a commutation money… He must pay a tax for his land (kharaj), from which the early Muslims were exempt; he must pay other exactions for the maintenance of the army, in which he cannot enlist even if he offers to render personal service instead of paying the poll tax; and he must show by humility of dress and behaviour that he belongs to a subject class. No non-Muslim can wear fine dresses, ride on horseback or carry arms; he must behave respectfully and submissively to every member of the dominant sect… The dhimmi is under certain legal disabilities with regard to testimony in law courts, protection under criminal law, and in marriage… He cannot erect new temples, and has to avoid any offensive publicity in the exercise of his worship… [In short], every device short of massacre in cold blood was resorted to in order to convert heathen subjects.13

The violent suspicion of Christians by Muslim authorities would reach its apex in the Armenian Genocide, in which one and a half million Armenians were slaughtered by the Ottoman Empire. With World War I in progress, the Ottoman Empire accused the Armenians of being in cahoots with Imperial Russia. This was used as a pretext to systematically exterminate the Armenian people, long distrusted as a Christian island subsisting within Islamic imperialism. The Armenian Genocide, enabled in part by German authorities in Turkey, would go on to inspire the Nazi holocaust of European Jews. To quote Adolf Hitler from his Obersalzberg Speech, addressed to Wehrmacht commanders a week before the German invasion of Poland:

I have placed my death-head formation in readiness – for the present only in the East – with orders to them to send to death mercilessly and without compassion, men, women, and children of Polish derivation and language. Only thus shall we gain the living space which we need. Who, after all, speaks today of the annihilation of the Armenians?14


The logic behind Ibn Taymiyyah’s annihilatory pronouncements flows primarily from the hadith literature. The justification for suicide missions is clear from the following hadith, recounting the story of a young man who ordered his own death to demonstrate the sovereignty of God. The abridged narration reads as follows:

So they [the king’s men] took him [the young man] and he said: “O Allah, save me from them and what they want to do.” It was quite soon that the boat turned over and they were drowned and he came walking to the king, and the king said to him: “What has happened to your companions?” He said: “Allah has saved me from them”, and he said to the king: “You cannot kill me until you do what I ask you to do.” And he said: “What is that?” He said: “You should gather people in a plain and tie me to the trunk of a tree. Then take hold of an arrow from the quiver and say: In the name of Allah, the Lord of the worlds; then shoot an arrow. If you do that, then you will be able to kill me.” So he (the king) called the people in an open plain and tied him (the boy) to the trunk of a tree, then he took hold of an arrow from his quiver and placed the arrow in the bow, and then said: “In the name of Allah, the Lord of the young boy.” He then shot an arrow and it struck his temple. He (the boy) placed his hands upon his temple and he died, and the people said: “We affirm our faith in the Lord of this young man.”15

As regards the persecution or wholesale slaughter of those who differ religiously, one need only point to the hadith narrated by Abu Said al-Khudri, describing the genocide of the Jewish Banu Quraizah tribe in 627 A.D. According to this hadith, on the orders of Muhammad, almost nine hundred Jews of Banu Quraizah were massacred by Muslims. The killing began early in the day, ending in torchlight. Those who escaped death were taken captive and sold at slave markets:

The people of (Banu) Quraizah agreed to accept the verdict of Sa’ad bin Muadh. So the Prophet sent for Sa’ad, and the latter came (riding) a donkey and when he approached the Mosque, the Prophet said to the Ansar, “Get up for your chief”, or “for the best among you.” Then the Prophet said (to Sa’ad), “These have agreed to accept your verdict.” Sa’ad said, “Kill their warriors and take their offspring as captives.” On that the Prophet said, “You have judged according to Allah’s Judgement”, or “the King’s Judgement.”16

A separate narration by Atiyyah al-Qurazi gives us an insight into the selection process:

I was among the captives of Banu Quraizah. They (the Companions) examined us, and those who had begun to grow (pubic) hair were killed, and those who had not were not killed. I was among those who had not grown hair.17

Indeed, one could cite innumerable examples of ISIS’ barbarity being justified by scripture. For now, however, it is sufficient to state that until the tradition of ijtihad is reborn in mainstream circles, the rulings of Ibn Taymiyyah will continue to inspire the most heinous of crimes. The impact of this puritan ignoramus on the development of mainstream Islam and Muslim society, whose collective instinct is to blame Western infidels for their own backwardness, cannot be understated. On that note, let us now delve briefly into the mindset of Salah ad-Din al-Ayyubi, known as ‘Saladin’ here in the West.



Part III

Saladin the Merciless

It is disturbingly common for Western academics to be duped by revisionist depictions of Islamic history. Take the Nobel laureate Amartya Sen, for example. In his book Identity and Violence, Sen actually cites Saladin, the bloodthirsty Muslim tyrant of the twelfth century, as an exemplar of pluralism and tolerance. To quote the writer Baha’uddin, who entered Saladin’s service in 1188 and was with him until his death:

He hated philosophers, heretics, materialists and all the opponents of the law. For this reason he commanded his son Al-Malik az-Zahir, prince of Aleppo, to punish a young man called As-Suhawardi, who called himself an enemy of the law and a heretic. His son had the man arrested for what he had heard of him and informed the sultan, who commanded that he be put to death. So he was killed and left hanging on the cross for several days.18

Saladin clearly had no interest in the plurality of beliefs, or in the exchange of ideas. Here is Baha’uddin again:

No sooner had he [returned] then a Frank, captured from the enemy army, was brought to him. He invited the man to embrace Islam, and when he refused, gave the order for his head to be cut off, which was done in his presence.19

We thus see that Saladin had no interest in the rights of those who reject faith. The historian Imad ad-Din quotes Saladin himself upon his conquest of Jerusalem:

“Neither amnesty nor mercy for you [the Christian peoples within Jerusalem]! Our only desire is to inflict perpetual subjugation upon you. Tomorrow will make us your masters by force. We shall kill and capture you wholesale, spill men’s blood and reduce the poor and the women to slavery.”20

Here is Imad ad-Din again on what happened to the women of Jerusalem:

Women and children together came to 8,000, and were quickly divided up among us, bringing a smile to Muslim faces at their lamentations. How many well-guarded women were profaned?… How many noblemen took them as concubines?… How many lovely women were the exclusive properties of one man? And how many great ladies were sold at low prices, and close ones set at a distance, and lofty ones abased, and savage ones captured, and those accustomed to thrones dragged down?21

In a letter to Caliph Nasser, after the death of Nur ad-Din’s son in 1181, Saladin claimed to be the true heir to the Zangid legacy, and thus espoused a grand imperial design extending well beyond the liberation of Jerusalem and the destruction of the Crusader states. Were he to be given possession of Mosul, Saladin hinted, this would lead to the capture of Jerusalem, Constantinople, Georgia, and North Africa “until the word of God is supreme and the Abbasid Caliphate has wiped the world clean, turning the churches into mosques.”22 Eight years later, in a conversation with his aide and biographer, Ibn Shaddad, Saladin reiterated his imperial dream:

“When God Almighty has enabled me to conquer the rest of the coast,” he said, “I shall divide up the land [among my sons], make my testament, then cross the sea to their islands to pursue them until there remains no one on the face of the earth who does not acknowledge Allah – or I die [in the attempt].”23


There are those who claim that “we can’t judge Saladin by the standards of our time.” But of course, if there is total incommensurability between our times and some remote time in the past, then not only can we not pass adverse moral judgements, we cannot pass favourable judgements either. If we can’t condemn Saladin for his actions, then we can’t praise him either. And yet, many relativists feel justified in praising the “Golden Age” of Islamic tolerance, thus judging by medieval standards of tolerance, all the while denouncing the behaviour of European imperialists as barbaric, thus judging by present-day standards of cruelty. For these people, moral relativism seems to be a matter of expediency.



References

1. From Babel to Dragomans: Interpreting the Middle East, page 375.

2. ‘Was It Inevitable?’ in Terrorism and the New War, Hoge and Rose, page 61.

3. Patricia Crone, Meccan Trade and the Rise of Islam, page 246.

4. Ibid, pages 247, 249-250.

5. Muhammad ibn Umar al-Waqidi, Kitab al-Maghazi, Volume 3, page 1113.

6. Patricia Crone, Meccan Trade and the Rise of Islam, pages 243-244.

7. ‘Islamic Conceptions of the Holy War’ in The Holy War, Thomas Patrick Murphy, pages 141-143.

8. Hamilton A.R. Gibb, Studies on the Civilization of Islam, pages 38-39.

9. Ibid, page 22.

10. The only exceptions to this rule were the Russian and the Austro-Hungarian empires.

11. Majmu’ al-Fatawa 28: 540.

12. Quoted in Taysir al-‘Az’z al-Hamid, Sulayman Ali ash-Shaykh.

13. History of Aurangzib, five volumes (1912-1924).

14. Louis Paul Lochner, What About Germany? (1942), pages 11-12.

15. Sahih Muslim 42: 7148.

16. Sahih Bukhari 5, 59: 447.

17. Sunan Abu Dawud 38: 4390.

18. Francesco Gabrieli, Arab Historians of the Crusades.

19. Ibid.

20. Ibid.

21. Ibid.

22. Lyons and Jackson, Saladin, pages 193-194.

23. Ibn Shaddad, Al-Nawadir al-Sultaniya, pages 24-25.



Pic 2

The conquest of Spain (Hispania) by the Umayyad Caliphate in the eighth century is a major source of inspiration for Islamic imperialists today.

A Short Speech on Islamism & the Far-Right (Audio)

The objective of Al-Qaeda is not the emancipation of the Palestinians, but the establishment of tyranny in the Muslim world by means of indiscriminate violence in the non-Muslim world, and those who confuse the two issues are idiots who don’t always have the excuse of stupidity.

Christopher Hitchens



What follows below, dear reader, is a speech I delivered at a recent branch meeting of Solidarity (AAA). This meeting was held at the office of Paul Murphy T.D. to discuss the growth of the far-Right in Europe and how the Left ought to respond to it.



Transcript (Extended):

I think the best way to respond to the rise of the far-Right is by trying to understand the appeal of these movements to ordinary people. When you look at the likes of Geert Wilders, undoubtedly, a huge part of their appeal is their willingness to criticise Islamism and to highlight the genuine problems with Muslim integration, however crude or sinister their approach may be.

People are increasingly fed up with being accused of racism or xenophobia for talking about these issues. They resent the nonsense charge of ‘Islamophobia’, which conflates criticism of Islam with anti-Muslim bigotry – two very different things.

They’re concerned about the development of Muslim-only neighbourhoods, where self-imposed “community leaders” make it their business to intimidate non-Muslims and to create a safe haven for extremism.

They’re worried about the rapid growth of Wahhabi-Salafi mosques and schools, where children and young adults are taught to establish the Islamic State and to regard the Christians and Jews as their mortal enemies.

They’re frightened by the widespread establishment of shari’a courts, which allow Muslim men to bypass secular law when it doesn’t suit them – particularly when it comes to spousal abuse and inheritance rights.

They’re appalled by the proliferation of Muslim grooming gangs who rape non-Muslim women, with the victims often being completely ignored by the police, who don’t want to be seen as racist.

These are all legitimate concerns, so when the Left summarily dismisses them, people are naturally drawn to the far-Right. When the Left refuses to take a principled stance against Islamic fascism, when we hesitate to defend the Enlightenment values that make our society possible (reason, humanism, universalism), then it falls to the Right to take up that mantle – even if these groups have nothing to do with the Enlightenment, but rather use it as a cover for racist bigotry.

I’m going to return to the issue of fascism for one minute. At one of the last branch meetings, I was told by a few people in the Socialist Party that I shouldn’t equate Islamism with fascism, that there’s a fundamental difference between the two. Well I don’t see it: in fact, I could go on for hours about the similarities between them:

Both movements are based on a cult of death that despises the intellect. Both are bitterly nostalgic for past empires, and seek to restore them through violence and the expulsion of undesirables. Both are obsessed with perceived “humiliations” and are thirsty for revenge. Both are chronically infected with anti-Semitic paranoia. Both are inclined to leader worship and to stressing the power of one great book. Both have a strong commitment to sexual repression and to subordination of the female. Both despise art and literature as symptoms of degeneracy; both burn books and destroy museums and treasures.

I could keep going with this, but I think it would be better to offer a way forward, so here’s what I propose: If the Left needs the help of ex-Muslims and liberal Muslims in combating Islamism – and I think it does – then that’s a good starting point: we are more than willing to help. But let’s do it sooner rather than later, because the Right seems to be growing exponentially, meaning we don’t have a whole lot of time.

Dispensing with Anti-GM Nonsense: The Creation of Fluorescent Protein Variants

True ignorance is not the absence of knowledge, but the refusal to acquire it.

Karl Popper



This article was prompted by a rather unpleasant exchange between myself and someone whose scientific knowledge is severely lacking. During this exchange, inevitably, the issue of genetic modification (GM) came up. When it did, I discovered that this individual, who is a sitting councillor for Limerick City North, opposes GM because he “doesn’t trust science under capitalism”. Such logic betrays not only a fundamental ignorance of what GM is, but also a conspiratorial mindset that is more befitting of Alex Jones and his acolytes..

Put simply, GM is a considerably more targeted version of artificial selection: we use it to make pest-resistant crops and life-saving recombinant protein drugs. Indeed, if I have type 1 diabetes, should I not take recombinant human insulin because we’re living under capitalism? The same applies to medicine more broadly: if I have a throat infection, should I not take antibiotics because we’re living under capitalism? What if I’m travelling to a country where polio is widespread: should I not get vaccinated because we don’t have a planned economy?

It should be stated, of course, that the profit motive is a major problem in R&D. Ben Goldacre and others have done an excellent job of exposing dubious practices in the pharmaceutical industry, for example. We should recognise, however, that this doesn’t mean the science itself is bad. We can fight for economic justice without alleging some vaguely evil conspiracy by geneticists, who have better things to be doing with their time. Indeed, if the reader wants some insight into the GM process, he can check out my final year project below. Alternatively, if the reader is short on time, GM can be summed up in seven easy steps:

1..We order a recombinant plasmid, i.e. a molecule of circular DNA into which our gene of interest has been inserted. For plant transformation, the most commonly used plasmid derives from the naturally occurring soil bacterium, Agrobacterium tumefaciens.

2..To confirm the identity of the plasmid, we induce its expression by growing it up in Luria Bertani broth. This broth contains ampicillin, meaning only plasmids designed with a resistance to ampicillin will survive.

3..Having purified our plasmid, we use a Miniprep Kit to break open the circular DNA. This is necessary to prepare the DNA for Polymerase Chain Reaction (PCR), which is our method of inducing the desired mutation.

4..In PCR, the double-stranded DNA is separated into two single strands. At this point, a pair of primers – short stretches of DNA – selectively bind to each strand. The forward primer, containing the altered nucleotide, binds to the bottom strand. The enzyme DNA Polymerase then synthesises new double-stranded DNA incorporating the mutation.

5..To confirm the success of mutagenesis, the new DNA is run on an agarose gel. It is then purified and ligated (sealed) to form a plasmid. Steps 2 to 5 are then repeated until all the desired mutations have been achieved.

6..The ligated plasmid is then mixed with plant cells or cut pieces of plants such as stems or leaves (explants). Some of the cells will take up the T-DNA, i.e. the section of the plasmid containing the mutated gene. The plasmid then inserts the T-DNA into one of the plant’s chromosomes, thus forming transgenic cells (GMOs).

7..The transgenic cells are then regenerated to form whole plants using tissue culture methods. The most viable samples are selected for the initiation of trial runs, which are performed in strict compliance with EPA regulations.

Using this method, we can modify corn to put up an effective resistance to Aspergillus, a mould that turns grains into health hazards. Through the induction of a few simple nucleotide changes, the corn cells are able to release specialised RNA molecules preventing the mould from releasing carcinogenic toxins. This is something which is only achievable through genetic engineering; there is only so much selective breeding can do. The same goes for recombinant protein drugs, which are produced by culturing E. coli cells transformed with plasmid mutants on a large-scale. Those who oppose GM ought to consider the plight of those with anaemia, who require recombinant erythropoietin (e.g. Epoetin alfa, manufactured by Amgen) to avoid chronic renal failure.

Picture0

A vial of recombinant human insulin, used to treat diabetes by lowering levels of glucose in the blood.


‘Generation, Expression & Purification of Fluorescent Protein Variants’

Kareem Muhssin, B.Sc.

Submission Date: April 2014


Abstract

Recombinant lectins are those whose glycan-binding properties have been altered by site-directed mutagenesis to recognise and selectively bind to specific glycan structures. By design, then, these lectins play an important role in the analysis of glycoprotein therapeutics, including the detection of glycosylation changes on the cell surface. The efficiency of these roles can be enhanced by fusing the lectins with fluorescent proteins such as EGFP and DsRed, for this enables the actual binding of glycans to be studied under fluorescence microscopy. By amplifying the fluorescence patterns of these fusion partners, we can optimise the visibility of labelled lectins; this is achieved by the induction of specific single-point mutations (SNPs). The induction of these SNPS, along with the induced expression of the resulting mutants and their purification by affinity chromatography, would comprise the bulk of the project.

In generating a palette of identical proteins with different emissions, we allow for more efficient labelling of lectins: for if the difference between the proteins is only a matter of two or three base-pairs, then any complications in the immobilisation of lectin-fluorescent protein fusions onto cell surfaces cannot be put down to differences in protein structure. In the author’s case, the parent protein was wild-type DsRed. The aim was to alter DsRed such that it emits green instead of red fluorescence; this protein is known as AG4. While the latter was not ultimately achieved, the author did obtain a much enhanced red mutant – known as E5up with V71M – which can now be used by the Irish Separation Science Cluster (ISSC) in their research. This research involves the detection of surface glycosylation changes – indicating apoptosis – in Chinese Hamster Ovary (CHO) cells under stress, and working to bring these cells back to a healthy state.

In producing a fluorescent protein with an enhanced emission pattern, the essential goal of the project was reached. In learning new scientific skills and refining existing ones, the broader goal was too.


List of Figures

Figure 1: The efficient immobilisation of an EGFP-lectin fusion onto the viscous surface area of a polyHIPE polymer

Figure 2: Flow diagram of the mutations used to obtain AG4 from DsRed

Figure 3: The GFP chromophore compared to its counterpart in DsRed

Figure 4: 1 kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel

Figure 5: Broad Range Protein Marker 2-212kDa visualised by Coomassie Blue staining on an SDS Polyacrylamide gel

Figure 6: Flow diagram of the physical work involved

Figure 7: Results of PCR confirmation gel for pQE-30, now carrying E5up

Figure 8: Results for transformation of concentrated JM109 with pQE-30 carrying E5up (enhanced red), viewed under the transilluminator

Figure 9: Contamination of GFP with DsRed (as expressed by JM109), viewed under the transilluminator

Figure 10: Results of PCR confirmation gel for pQE-30, presumed to be carrying E5

Figure 11: Results of 40-minute PCR confirmation gel for pQE-30, with twice the number of samples

Figure 12: Results of PCR confirmation gel for pQE-30, presumably now carrying E5

Figure 13: Results for transformation of JM109 with pQE-30 carrying E5 (enhanced red), viewed under the transilluminator

Figure 14: The results of agarose gel electrophoresis when the agarose hasn’t set properly

Figure 15: The amino acid sequence of DsRed-E5up, showing the replacement of Valine with Alanine at position 105

Figure 16: Cultures of JM109 expressing M3 Green (right) and M3 Red (left) in LBA, viewed under the transilluminator

Figure 17: Distorted SDS Polyacrylamide gel

Figure 18: M3 Red protein (presumed to be E5up, E5, or E57) after purification by immobilised nickel affinity chromatography

Figure 19: The results of SDS-PAGE for our M3 Red elutions, the filtered lysate, the unbound lysate, and the imidazole washes

Figure 20: Absorbance readings for our buffer-exchanged M3 Red elutions

Figure 21: Our M3 Red elution compared to presumed E57 under UV light

Figure 22: Results of a 0.7% agarose gel to confirm the presence of pQE-30 carrying M3 Red and pQE-30 carrying M3 Green

Figure 23: M3 Red (super-enhanced DsRed) compared to wild-type DsRed under the transilluminator

Figure 24: Fluorescence excitation and emission spectra of matured DsRed (~2 weeks at 25°C) compared to E5up

Figure 25: Absorbance readings for original DsRed elutions

Figure 26: Absorbance readings for our second series of M3 Red elutions

Figure 27: Results of SDS-PAGE for neat M3 Red and dilutions to 1 in 6

Figure 28: The amino acid sequence for M3 Red, demonstrating the induction of V71M and V105A, but not S197T

Figure 29: The amino acid sequence for our second mutant, showing the induction of V71M instead of S197T

Figure 30: Part of the amino acid sequence for M3 Green, demonstrating the induction of V71M and V105A, but not S197T

Figure 31: The amino acid sequence for M3 Red, with special emphasis on the residues preceding the Methionine at position 71

Figure 32: Part of the amino sequence for M3 Green, showing random mutations at positions 67-70

Figure 33: The complete amino acid sequence for M3 Green, divided across two reading frames

Figure 34: The results of SDS-PAGE for our M3 Red elutions


List of Tables

Table 1: The various mutants of DsRed, their phenotypic differences, and the mutations required to obtain them

Table 2: The three areas where DsRed needs improvement before it can be used for lectin fusions, compared to GFP


Table of Contents

Chapter 1: Introduction

  • 1.1: The generation of fluorescent protein variants for use in the early detection of apoptosis
  • 1.2: The immobilisation of lectin-fluorescent protein fusions onto cell surfaces
  • 1.3: One example of the use of recombinant lectins in the analysis and purification of biotherapeutics
  • 1.4: The various mutants of DsRed and the structure of the DsRed chromophore
  • 1.5: Two short notes on site-directed mutagenesis

Chapter 2: Materials & Methods

  • 2.1: Making up Luria Bertani (LB) broth / agar
  • 2.2: Making up LB amp (LBA)
  • 2.3: Preparing LBA agar plates
  • 2.4: Preparing overnight cultures of KRX and JM109
  • 2.5: Culturing plasmid DNA / E. coli expressing plasmid DNA
  • 2.6: Plasmid DNA isolation
  • 2.7: Agarose gel electrophoresis
  • 2.8: Making up TE Buffer
  • 2.9: Preparing glycerol stocks
  • 2.10: Preparation of mutagenic primers for PCR
  • 2.11: Mutagenesis PCR
  • 2.12: DNA purification from PCR mixtures
  • 2.13: Restriction digest
  • 2.14: Making up Terrific Broth (TB)
  • 2.15: Making up Super Optimal Broth (SOB)
  • 2.16: Making up SOB with catabolite repression (SOC)
  • 2.17: Running a SyBr Safe gel
  • 2.18: DNA purification from agarose gels
  • 2.19: Ligation
  • 2.20: Growing up competent cells of KRX and JM109
  • 2.21: Transformation of competent cells
  • 2.22: Streak plates
  • 2.23: Sending plasmid DNA for sequencing
  • 2.24: Preparing overnight cultures in TBA
  • 2.25: Preparing an SDS Polyacrylamide gel
  • 2.26: SDS-PAGE, staining & de-staining
  • 2.27: Inducing protein expression
  • 2.28: Making up lysis buffer
  • 2.29: Stripping and recharging the IMAC resin
  • 2.30: Cell lysis to obtain recombinant protein
  • 2.31: IMAC
  • 2.32: Buffer exchange with PBS (protein purification)

Chapter 3: Diary of Work Done, Results & Data Interpretation

  • 3.1: Prelude
  • 3.2: Diary
  • 3.21 (Days 1–9): The induction of V105A and the expression of E5up
  • 3.22 (Days 9–23): The induction of S197T and the expression of E5
  • 3.23 (Days 23–28): The induction of V71M and the expression of AG4
  • 3.24 (Days 28–35): Protein expression analysis & induced expression
  • 3.25 (Days 35–47): Protein purification by nickel affinity chromatography

Closing Statement

Acknowledgements

References

Appendices

  • Appendix A: The amino acid sequence of DsRed
  • Appendix B: The nucleotide sequences of our mutagenic primers
  • Appendix C: Table of the amino acids and their respective codons
  • Appendix D: Diagram of the mutagenesis process and primer design

Chapter 1: Introduction

In the literature review, we concerned ourselves with the process of glycan evolution and how glycosylation can be used to enhance the efficacies of protein drugs. For the project, our focus was the use of site-directed mutagenesis to obtain enhanced variants of a wild-type fluorescent protein. These variants would be used by researchers at the ISSC in the early detection of apoptosis in eukaryotic cells. The onset of apoptosis is indicated by specific changes in glycosylation on the cell surface; thus, by fusing these fluorescent variants with lectins that bind to extracellular glycans, these changes can be monitored under fluorescence microscopy. The importance of glycosylation, therefore, was the ultimate bridge between the literature review and the project.

1.1: The generation of fluorescent protein variants for use in the early detection of apoptosis

Fluorescent proteins can be defined as structural homologues of the green fluorescent protein expressed by Aequorea victoria, a bioluminescent hydrozoan jellyfish, that share the ability to form a visible internal wavelength fluorophore from a sequence of three amino acids within their own polypeptide sequence. Fluorescent proteins are most commonly used for studying the dynamics and localisation of specific organelles (Rizzuto et al., 1995) or recombinant proteins (Ballestrem et al., 1998) in living cells. With regards to the latter, DsRed, a red fluorescent protein expressed by the Discosoma species of mushroom anemone, has proven especially useful.

The central aim of the project was thus to produce a series of DsRed variants with enhanced fluorescence patterns, by the successful induction of specific SNPs. We would achieve this by performing site-directed mutagenesis, inducing the expression of the resulting mutants and purifying them by Immobilised Metal Affinity Chromatography (IMAC). These fluorescent variants would then be used by researchers at the ISSC to create fusions with novel recombinant lectins, with the aim of detecting stress-induced glycosylation changes in CHO cells. In this endeavour, after the tagged cells have been treated with different stresses, flow cytometry is employed to detect any resultant changes in lectin binding.

In flow cytometry, lasers can be set for different wavelengths; therefore, the advantage of having mutants with different emissions is enormous. This is especially true if we want to label two glycoprotein drugs at the same time, or perform sequential labelling. The advantage is even greater when these mutants only differ by a few base-pairs, as would be the case with our DsRed mutants. Although our proteins would ultimately exhibit different fluorescences, the difference between them would only amount to two base-pairs, thus making them virtually the same. This allows for more effective labelling of lectins: for when obtaining data using flow cytometry, the possibility of interference or overlapping between different fluorescent proteins is always a major concern.

Apoptosis is a natural response in cells that are under stress, whether it is the stress of uncontrolled differentiation in cancer or the shear stress experienced by cells in a bioreactor. The onset of apoptosis is indicated by certain changes in glycosylation on the cell surface – for example, a decreased expression of O-glycosylated proteins (Batisse et al., 2004) and/or a marked decrease in cell surface sialylation (Shiratsuchi et al., 2002). Therefore, the ability to detect these changes early on is crucial in attempting to restore the cells to a healthy state (by altering the cell culture conditions). We can do exactly that by fusing recombinant lectins with fluorescent proteins and then immobilising these fusions onto the surfaces of cells to monitor any changes in lectin binding.

The importance of such an early detection method for the biopharmaceutical industry is obvious: for if we have a 10,000 litre vat of cells, and we want to determine if these cells are beginning to become stressed, then we need to so before the batch is compromised. At the moment, we have no choice but to take out a sample of cells, release the glycans, perform a mass-spec, and purify them by HPLC. It takes approximately 14 hours to carry this process out, by which time the cells are likely to be in trouble. There is no wonder, then, why so much focus is being put on developing a detection method that is fast and powerful – including the efforts of the ISSC.

1.2: The immobilisation of lectin-fluorescent protein fusions onto polymer and cell surfaces

Strictly speaking, fluorescent labelling is defined as the covalent attachment of a fluorophore to another molecule, such as a protein or nucleic acid. This is generally accomplished using a reactive derivative of the fluorophore that selectively binds to a functional group present on the surface of the target protein. Alternatively, recombinant DNA technology may be used to generate a fusion between the target protein and a naturally-fluorescent protein such as GFP or DsRed.

When we immobilise lectins, enzymes and other proteins onto the surfaces of cells, one of the easiest ways to check the efficiency of this immobilisation is via their fusion with fluorescent proteins. The likes of GFP and DsRed have the ideal amount and distribution of surface lysine residues for binding to a variety of chemically-activated surfaces. In addition, the fusion process itself is relatively simple to perform, involving the creation of a construct containing the fluorescent protein at either the NH2−terminus or the COOH-terminus of the protein of interest and the insertion of this construct into an appropriate expression vector, which is accomplished by ligation into a multi-cloning site. To quote Snapp et al. (2005) on the final design of the construct:

The final FFP [fused fluorescent protein] construct will contain an in-frame fusion between the FP and the protein of interest, an unambiguous initiating methionine within the appropriate Kozak sequence (for eukaryotes, this is 5′-ACCATGG-3′, where the internal ATG is the initiating methionine), a linker between the FP and the protein of interest (if necessary), and appropriate regulatory elements.

There are three bonuses to fusing lectins with fluorescent proteins:

1. By virtue of being visible, these proteins can give us a direct measurement of the efficiency of lectin immobilisation. This is determined quantitatively by the percentage of the fluorescent protein that has bound to the surface, and if this protein is evenly distributed. Figure 1 displays the immobilisation of an EGFP-lectin fusion onto the surface of a PolyHIPE polymer under 10X microscopy. PolyHIPEs are porous polymers with highly viscous surface areas that enable their use in reaction supports, separation membranes and other applications.

Picture1

Figure 1: The efficient immobilisation of an EGFP-lectin fusion onto the viscous surface area of a PolyHIPE polymer. Image sourced from Audouin et al. (2012): ‘Polypeptide grafted macroporous PolyHIPE by surface initiated N-Carboxyanhydride Polymerization as a platform for bioconjugation’. Macromolecules, 45, 6127-6135.

In the diagram, we can see a nice distribution of the fluorescent protein, indicating a high efficiency of lectin immobilisation onto the polymer surface. This efficiency tells us that we don’t have to label each lectin in a different way: rather, we now have a generic way to do so.

2. Fluorescent proteins have often been found to stabilise certain lectins.

3. Orientation-specific immobilisation becomes more possible, as when we immobilise a fluorescent protein partner onto a particular lectin, we are making sure that the lectin binding pocket is now orientated correctly. (The protein is always fused to an N- or C-terminus, so it doesn’t interfere with the binding pocket.)

1.3: One example of the use of recombinant lectins in the analysis and purification of biotherapeutics

In the production of biopharmaceuticals, the maximum possible level of purity is desirable. By labelling our protein of interest with an affinity tag – be it a short peptide sequence or a full protein – we can precipitate the final product from the thousands of contaminants involved with relative ease. For the analysis of glycoprotein therapeutics, labelling with recombinant lectins – sometimes fused to fluorescent proteins – is considered the most suitable option. Indeed, the novel recombinant lectins developed by the ISSC are much better than commercial lectins at binding to specific targeted therapeutics.

One example of a recombinant lectin that has proven useful in the analysis and purification of biotherapeutics would be the PA-IL lectin, expressed by Pseudomonas aeruginosa. As part of a recent collaborative study by the ISSC and Dublin City University (DCU), specific amino acid residues located in the carbohydrate binding site of a recombinant PA-IL protein (rPA-ILNmE6) were substituted at random by site-directed mutagenesis (Keogh et al., 2014). This resulted in high affinity and specificity of the protein for N-acetyl-lactosamine (LacNAc) and for β-linked galactose exhibited by N-linked glycans on the surfaces of glycoproteins (Keogh et al., 2014).

The researchers demonstrated the use of this novel recombinant prokaryotic lectin in the analysis of glycoproteins and in the isolation and selective fractionation (purification) of glycoproteins and their glycoforms. One way of doing this involved immobilisation of the lectin onto magnetic particles (Keogh et al., 2014). To quote from the paper:

To evaluate the ability of these lectin functionalized magnetic particles to isolate glycoproteins displaying terminal β1–4 galactose, pull down assays were performed in 1.5mL tubes, using a test protein mixture prepared by mixing asialotransferrin with recombinant green fluorescent protein (GFP) and RNase B. Fractions of unbound and bound protein were ultimately evaluated by SDS-PAGE. [From this gel], it could be clearly seen that the rPA-ILNmE6 magnetic beads selectively and efficiently extracted the asialotransferrin from the protein mixture.

1.4: The various mutants of DsRed and the structure of the DsRed chromophore

DsRed was first discovered in late 1999 by a research team of the Russian Academy of Science, who observed that reef Anthozoa express fluorescent proteins with hues ranging from red to cyan (Matz et al., 1999). Soon afterwards, wild-type DsRed was found to have certain properties that prevent its efficient use as an in vivo reporter. These drawbacks, along with obtaining a palette of DsRed mutants to work with, are what necessitate site-directed mutagenesis. They include the following:

1. When compared to wild-type GFP, Baird et al. (2000) and Terskikh et al. (2001) observed a slow rate of fluorescence maturation for DsRed. This difference is most striking at lower temperatures (just below room temperature) – temperatures at which GFP has maximum folding efficiency (Kimata et al., 1997). This limits the utility of DsRed for studies of heterologous expression systems that are maintained at low temperature (such as Drosophila or Xenopus).

In contrast, DsRed and all other Anthozoa proteins studied thus far mature faster than GFP at temperatures higher than 30°C (Terskikh et al., 2001). These temperature-dependent rates could be evolutionary adaptations to the animals’ natural habitat: corals live in the warm Indo-Pacific waters, while Aequorea victoria lives in the cold boreal waters (Terskikh et al., 2001).

2. Even after lengthy periods of maturation (several weeks), wild-type DsRed retains residual green fluorescence at 500 nm, amounting to 4–5% of the major red fluorescence peak (Terskikh et al., 2001). This can be a major source of problems in multicolour detection applications.

Both of these issues can be overcome by the successful induction of specific SNPs. By the induction of V105A in wild-type DsRed, we can accelerate fluorophore maturation such that not only is the red fluorescence enhanced, but any residual green fluorescence is eliminated entirely. Presumably, substituting a bulky Valine for a more compact Alanine causes the overall structure to relax, facilitating the complete maturation of the red fluorophore (Terskikh et al., 2001). The resulting mutant is known as E5up.

It is also possible to obtain DsRed mutants exhibiting intense green fluorescence, if so desired. One such mutant, AG4, can be achieved by the induction of V71M in E5 (i.e. DsRed carrying both V105A and S197T). The induction of S197T enhances green fluorescence; the induction of V71M abrogates the enhanced red of V105A, thus enabling the DsRed mutant to fluoresce green. Most likely, the introduction of a larger side chain that comes with substituting Methionine for Valine produces substantial distortion of the fluorophore environment, preventing complete maturation of the red fluorescence (Terskikh et al., 2001).

Figure 2 represents how AG4 can be obtained from DsRed by the successive induction of V105A, S197T and V71M:

Picture2

Figure 2: Flow diagram of the mutations used to obtain AG4 from DsRed.

The author of this report opted to obtain AG4: for while the production of yet another green fluorescent protein may not seem to be of any advantage initially, in reality, it can be used as an ideal counterpart for DsRed in the multi-coloured labelling of novel recombinant lectins. The minimal sequence differences between AG4 and DsRed ensure their similar behaviour on cell surfaces, i.e. precipitation, non-specific accumulation, and interaction with cellular components (Terskikh et al., 2001). Thus, any observable differences in the behaviour of two proteins – one labelled with DsRed, the other with AG4 – should be attributed to the proteins themselves, and not the fluorescent tags. Featured below is a table of the DsRed mutants and their phenotypic properties:

Table 1

Table 1: The various mutants of DsRed, their phenotypic differences, and the mutations required to obtain them.

It is important that we elaborate somewhat on the structure of the DsRed chromophore, for it is by site-directed mutagenesis in and around the chromophore that the red fluorescence is altered. Fluorescent proteins with different emission patterns amount to proteins with different chromophore environments.

The DsRed chromophore is an auto-catalytically produced variant of the GFP chromophore. As shown in Figure 3, the main difference between the two is that the DsRed chromophore has an additional double bond (drawn in yellow), which extends its conjugation and causes the shift to red fluorescence. Robert E. Campbell of the University of Alberta provides a more detailed explanation in this regard (2007):

The key difference between the red and green proteins is that Discosoma red fluorescent protein undergoes an additional fourth step in the chromophore maturation pathway, oxidizing the adjacent Cα-N bond to form an acylimine moiety that extends the conjugated system by two double bonds.

Picture3

Figure 3: One double bond – the result of pi-electron conjugation – is primarily what distinguishes the DsRed chromophore from that of GFP. Featured here are simplified versions of the structures of O. Shimomura (1979): ‘Structure of the chromophore of Aequorea green fluorescent protein.’ FEBS Letters 104.

In October 2000, Gross et al. of the University of Pennsylvania investigated the structure of the DsRed chromophore. From this study, they were able to identify key differences between the major properties of wild-type DsRed and wild-type GFP. Of these properties, the authors singled out three areas where DsRed needs improvement before it can be used as an efficient fusion partner: namely, suppressing its aggregative tendencies, and increasing both the completeness and the speed of maturation to the desired red form (Gross et al., 2000). These are summarised in the table below:

Table 2

Table 2: The three areas where DsRed needs improvement before it can be used for lectin fusions, compared to GFP. Abridged version of the table drawn up by Gross et al. (2000): ‘The structure of the chromophore within DsRed, a red fluorescent protein from coral.’ PNAS, vol. 97 no. 22, 11990–11995.

With regards to the extent and speed of chromophore maturation, although the residual green fluorescence in wild-type DsRed does not show up in the emission spectrum – due to fluorescence resonance energy transfer (FRET) within the tetramer – its excitation peak will nonetheless contribute spectral cross-talk in experiments that use GFP as a separate label or FRET donor (Gross et al., 2000). Therefore, if DsRed is to be used as a fluorescent label, then increasing the speed and the extent of chromophore maturation – thereby eliminating residual green fluorescence – is of crucial importance. By the induction of V105A, this is what we had hoped to achieve in the project.

1.5: Two short notes on site-directed mutagenesis

Site-directed mutagenesis is a convenient method for inducing a specific mutation at a specific site in a sequence of plasmid DNA. This mutation can be a substitution, deletion or insertion. Site-directed mutagenesis has many uses, such as the removal of restriction sites from a plasmid, investigating the function of specific amino acids in an enzyme, or amplifying the phenotypic properties of a particular protein.

With regards to PCR-based site directed mutagenesis, the principle is to design a pair of PCR primers adjacent to one other, such that it is the entire plasmid that is amplified by PCR. One of these primers – the forward primer – incorporates the desired mutation. The result of the PCR is a linear product whose ends can then be sealed together (after phosphorylation) with DNA ligase. The ligated vector is then taken up and expressed by E. coli (transformation). This process can be summarised in seven steps:

Step 1: Primer Design

Step 2: PCR

Step 3: Purification of the PCR product

Step 4: Phosphorylation of the 5’ termini (if necessary)

Step 5: Ligation of the DNA ends

Step 6: Transformation of competent E. coli

Step 7: Confirmation of success by DNA sequencing.

Short notes on primer design are included in Section 3.1 of this report. A detailed diagram on primer design and the mutagenesis process is also included in Appendix D.


Chapter 2: Materials & Methods

2.1: Making up Luria Bertani (LB) broth / agar

Prepare the required volume of distilled water in a beaker. Place the beaker on a heat-stir, with a magnet bar resting at the bottom. Set the heat-stir to the second-lowest setting. Weigh out the appropriate amounts of Tryptone, NaCl and Yeast Extract and add them to the beaker. (For making any volume of LB, these components are added at concentrations of 10 g/L.) Once the solution is homogenous, pour it into a graduated cylinder and top up to the correct volume with distilled water. Pour this into a duran that has been clearly labelled and sealed with autoclave tape. If making LB agar, it is at this point that agarose is added (Scharlau Agar Bacteriological 07-004) at a concentration of 15 g/L. The agarose is allowed to settle at the bottom of the jar.

Autoclave the jar, keeping the lid loose to ensure the LB is heated properly. (Check the volume of water in the autoclave before proceeding. Once the lid is shut, switch on the device and set the knob to steam.) The heating process takes approximately 30 minutes. Once the pressure has fallen below half a bar, set the knob to ‘exhaust’ and wait for the pressure to hit zero. At this point, open the lid and retrieve the LB. Once the autoclaved LB has cooled down (usually after 30-40 minutes), it is ready for use.

2.2: Making up LB amp (LBA)

Prepare a 100 mg/mL concentration of ampicillin in a sterilin tube (e.g. 0.359 g ampicillin in 3.59 mL distilled water). Aliquot 500 μL volumes of this preparation into a series of microfuge tubes. To make LB amp, add ampicillin to LB at a concentration of 1 μL/mL. (It is good scientific practice to do this in the fume hood.) The LBA is now ready for use.

2.3: Preparing LBA agar plates

In the fume hood, prepare a series of empty plates in close proximity to the Bunsen flame. Slowly and gently pour the LBA agar into each plate, making sure to cover each plate’s surface area. Keep the plates by the flame until they have settled, with the lids partially covering each plate. Close the plates once the agar has settled. The plates are now ready for use.

2.4: Preparing overnight cultures of KRX and JM109

Culture KRX and JM109 (ordered from PROMEGA) by inoculating 10 μL of each sample in two sterilin tubes of 5 mL LB. Prepare another tube of sterile 5 mL LB as a control. (It is good scientific practice to do all of this in the fume hood.) Store the tubes overnight at 37˚C in an incubator shaker. The following day, retrieve the tubes and examine their appearance. Successfully inoculated samples appear murky in colour. A cloudy appearance for LB (or LBA) that has not been inoculated indicates contamination.

2.5: Culturing plasmid DNA / E. coli expressing plasmid DNA

Add 10 μL of plasmid (or a single colony expressing the plasmid) to 5 mL LBA in a sterilin tube. Incubate overnight at 37˚C in an incubator shaker. The following day, retrieve the tubes and examine their appearance.

2.6: Plasmid DNA isolation

Sigma GenElute Plasmid Miniprep Kit

Transfer 1.5 mL of cell culture (expressing the plasmid) to a microfuge tube. Centrifuge the tube at 13,000 rpm for 2 minutes to pellet the cells. Remove the supernatant and resuspend the pellet in 200 μL of resuspension solution (kept in the fridge, as it contains RNase A solution). Add 200 μL of lysis solution. Gently invert the tube and leave to incubate at room temperature for 5 minutes. (Harsh mixing may cause the co-purification of unwanted chromosomal DNA.) Add 350 μL of neutralisation solution and mix by inversion to precipitate cell debris, lipids, proteins and chromosomal DNA. Leave the mixture at room temperature for 10 minutes. Collect the precipitate by centrifugation at 13,000 rpm for 10 minutes.

Prepare a spin column for binding by adding 500 μL of column preparation solution and centrifuging at 13,000 rpm for 1 minute. Transfer the supernatant to the prepared spin column and centrifuge for 30 seconds to bind the plasmid DNA. Discard the flow-through and add 750 μL of wash solution. Centrifuge at 13,000 rpm for 30 seconds to remove any contaminants. Discard the flow-through and dry the matrix by centrifuging at 13,000 rpm for 2 minutes. Transfer the spin column to a new microfuge tube and add 100 μL of distilled water. Elute the plasmid DNA by centrifugation at 13,000 rpm for 30 seconds.

2.7: Agarose gel electrophoresis

Prepare a 0.7% agarose solution by adding 0.7% agarose to TAE buffer and dissolving by boiling. The agarose is stored at 60˚C to prevent solidification. When required, the heated agarose is poured into plastic trays and allowed to set containing a plastic comb to form wells. 1X TAE buffer is used as the running buffer. Typically, 2 μL of gel loading dye is mixed with 7 μL of sample to help the sample settle in the well. A 1kb DNA ladder is used:

Picture4.png

Figure 4: 1 kb DNA Ladder visualized by ethidium bromide staining on a 0.8% TAE agarose gel. © New England Biolabs Inc.

The ladder is also loaded with gel loading dye. Gels are run at 120 volts for 20-40 minutes. The gel is then stained for 15 minutes by immersion in an ethidium bromide solution (wearing gloves, as ethidium bromide is carcinogenic). Gels are visualised using the UV transilluminator. Dispose of gels and gloves in the nearest ethidium bromide waste bin.

2.8: Making up TE Buffer

TE Buffer consists of a 10mM concentration of Tris-HCl and a 1mM concentration of Na2EDTA in 100 mL of distilled water. Calculate the amount in grams needed for each concentration, weigh them out, and add them to a duran of 100 mL distilled water. Using a dropper, adjust the pH to 8.0 with HCl. The TE Buffer is now ready for use. Example:

Tris-HCl has a molecular weight of 121.14 g/M. Therefore, for a 10mM concentration, weigh out 1.21 g. The working assumption here is a 1L volume; thus, for 100 mL, divide by 10 once more to get 0.121 g.

2.9: Preparing glycerol stocks

In the fume hood, prepare cryogenic vials in duplicate, each containing 0.5 mL of glycerol and 1 mL of competent cells in LBA / plasmid in LBA. Store the vials at -80˚C.

2.10: Preparation of mutagenic primers for PCR

The forward and reverse primers for each mutation are resuspended in the appropriate volumes of TE Buffer (amount of oligo X 10). Prepare 1 in 10 dilutions of these stocks (10 μL stock + 90 μL TE Buffer) in clearly labelled microfuge tubes. Use the stickers provided with the information sheet for each primer to label the tubes.

2.11: Mutagenesis PCR

PCR reactions are performed in the Veriti 96 well Thermal Cycler (Applied Biosciences). The reaction components are as follows:

Template DNA                        1 μL

dNTPs (10 μM)                        1 μL

Primers (10 μM)                     1 μL of each

Buffer (5X)                              10 μL                           Q5 High Fidelity

Distilled water                        35 μL

DNA polymerase                    1 μL                             Q5 High Fidelity

Prepare a master mix containing 4X each component for the four potential annealing temperatures (55, 60, 65, 70˚C). (The dNTP mix contains 5 μL of each dNTP in 30 μL of distilled water. The DNA polymerase is added last.) Aliquot 50 μL of the master mix into 4 PCR tubes each. Pulse the tubes to ensure the reaction is concentrated at the bottom. Program the Veriti 96 well Thermal Cycler as follows:

Browse/New methods → 2 minute extension → View/edit → Zones → Change to 3 minute extension → Run (check 50 μL) → Start PCR.

The 5X Buffer and DNA polymerase are stored at -20˚C.

2.12: DNA purification from PCR mixtures

After running a confirmation gel (5 μL for each PCR reaction), there should be 45 μL remaining in each PCR tube. Combine the samples that appear on the gel for 90 μL in one microfuge tube. Add 500 μL of capture buffer type 3 and mix the sample thoroughly by vortexing. Incubate the tube at 55˚C for 10-15 minutes until the agarose gel is fully dissolved. During this incubation, the tube is inverted every 2-3 minutes. Place the sample in a spin column and centrifuge at 13,000 rpm for 30 seconds. Discard the flow-through. Add 500 μL of wash buffer type 1 and centrifuge at 13,000 rpm for 30 seconds. Discard the flow through and dry the column in a new microfuge tube by centrifugation at 13,000 rpm for 2 minutes. Add 30 μL of distilled water to the centre of the column matrix and leave at room temperature for 2 minutes. Centrifuge the column at 13,000 rpm for 30 seconds to elute the DNA. The purified DNA is then stored at -20˚C.

2.13: Restriction digest

For a total of volume of 50 μL, prepare each reaction as follows:

Buffer (10X)                  5 μL                 Buffer 4

Purified plasmid          30 μL               Linear PCR product

Enzyme (DpnI)              1.5 μL              20,000 units/mL

Distilled water              13.5 μL

The restriction enzyme is added last. The reactions are incubated at 37˚C for 2 hours.

2.14: Making up Terrific Broth (TB)

For a 1L volume of TB, prepare the following in 900 mL of distilled water:

Tryptone                      12 g                 (Scharlau Casein Trypsic Peptone 07-119)

Yeast extract               24 g                 (Scharlau Yeast Extract 07-079)

Glycerol (50%)             4 mL

Autoclave the prepared mixture. After cooling, add 100 mL of 1M potassium phosphate buffer aseptically. The TB is now ready for use. (Some of the phosphate buffer may aggregate to form crystals. It is easy to mistake this as contamination.)

2.15: Making up Super Optimal Broth (SOB)

Prepare the following in the appropriate amount of distilled water:

Tryptone                      20 g/L              (Scharlau Casein Trypsic Peptone 07-119)

Yeast extract               5 g/L                (Scharlau Yeast Extract 07-079)

NaCl                               500 mg/L

KCl                                 2.5 mM

The molecular weight of KCl is 74.55 g/M; therefore, for a 0.0025M concentration, we need 0.1864g. The working assumption here is 1L, so for 500 mL of SOB (for example), divide by 2 to get 0.0932g. Add this amount to 500 mL of distilled water and autoclave.

Once the SOB has cooled, we need to add 2 mL of MgCl2 and MgSO4, at concentrations of 1M. (The molecular weight of MgCl2 is 95.22 g/M. The working assumption is 1L, so if we make up 20 mL, for example, we divide by 50 to get 1.9044g. Do the same for MgSO4.) Once these have been added, the SOB is ready for use.

2.16: Making up SOB with catabolite repression (SOC)

In the fume-hood, add 1 mL of filter-sterilised MgCl2 and MgSO4 to 100 mL of SOB. Proceed to add 2 mL of filter-sterilised glucose (50%). The SOC is now ready for use.

2.17: Running a SyBr Safe gel

Prepare a 0.7% agarose gel as normal. 3 μL of SyBr Safe is added to the gel before it sets. Add 8 μL of loading dye to each 50 μL digest. Once the gel has set, remove the comb and load 25 μL each of these samples. Run the gel at 120 volts for 20-40 minutes. View the gel under the transilluminator to identify the best expressed plasmid.

2.18: DNA purification from agarose gels

The DNA band to be purified from the SyBr Safe gel is excised using a scalpel. 500 μL of capture buffer type 3 is added and the sample is mixed thoroughly by vortexing. The tube is incubated at 55˚C for 10-15 minutes until the agarose gel is fully dissolved. During this incubation, the tube is inverted every 2-3 minutes. The sample is placed in a spin column and centrifuged at 13,000 rpm for 30 seconds. The flow-through is discarded. 500 μL of wash buffer type 1 is added and centrifuged at 13,000 rpm for 30 seconds. The flow-through is discarded, and the column is dried in a new microfuge tube by centrifugation at 13,000 rpm for 2 minutes. 30 μL of distilled water is added to the centre of the column matrix and left at room temperature for 2 minutes. The column is then centrifuged at 13,000 rpm for 30 seconds to elute the DNA. The purified DNA is then stored at -20˚C.

2.19: Ligation

For a total of volume of 50 μL, prepare each reaction as follows:

Purified plasmid        40 μL

Buffer (10X)                5 μL                 T4 DNA ligase buffer

Ligase (T4)                  2 μL                  T4 DNA ligase

Distilled water           3 μL

Pulse each tube, and incubate at room temperature for 3 hours.

2.20: Growing up competent cells of KRX and JM109

Add 2 mL of KRX and JM109 overnight cultures to 200 mL of SOB (to which 2 mL of MgCl2 and MgSO4 have been added). Incubate for roughly 3 hours in an incubator shaker. Measure the absorbance at 1.5 hour intervals, with the spectrophotometer set to 600 nm. Once the absorbance has reached 0.4 – 0.6, pour each culture into sterile centrifuge bottles and centrifuge at 4,500 rpm for 5 minutes to pellet the cells. Decant the supernatant and resuspend the cells in 80 mL of cooled TB buffer. Leave the resuspended solution on ice for 10 minutes. Pellet the cells by centrifugation at 5,500 rpm for 5 minutes. Decant the supernatant, and gently resuspend the cells in 15 mL of chilled TB buffer. Slowly add 7% DMSO, incubate the suspension on ice for 10 minutes. Prepare aliquots of 400 μL in sterile 1.5 mL microfuge tubes, and flash freeze them using a block that has previously been cooled to -80˚C. The competent cell aliquots, now ready for use, are then stored at -80˚C.

2.21: Transformation of competent cells

The aliquots of competent cells are thawed on ice. 200 μL of competent cells are gently mixed with 5 μL of plasmid DNA in a sterile microfuge tube (fume-hood). The tube is incubated on ice for 30 minutes to allow the DNA and cells to bind. The cells are heat-shocked at 42˚C for 30 seconds and put back on ice for 2 minutes. 800 μL of SOC broth is added to the cells (fume-hood) and they are incubated at 37˚C for 1 hour. 200 μL of this neat suspension is pipetted on to an LBA agar plate and is incubated overnight at 37˚C. The remaining 800 μL is centrifuged at 5,000 rpm for 60 seconds. 600 μL of supernatant is removed; the pellet is resuspended in the remaining 200 μL and spread onto an LBA agar plate, to be incubated overnight at 37˚C. A control plate is also prepared using 200 μL of competent cells that haven’t taken up the plasmid.

2.22: Streak plates

Sterilize the inoculation loop by passing it through a flame. When the loop has cooled, use it to pick a single colony of E. coli. Drag the inoculation loop across the edges of the agar surface until roughly 30% of the plate has been covered. Re-sterilize the loop each time, and rotate the plate 90 degrees. Drag the loop in a zig-zag pattern from the final streaked area towards the starting point, but do not connect the two. Incubate the plates overnight at 37˚C.

2.23: Sending plasmid DNA for sequencing

Aliquot 15 μL of purified plasmid DNA into two microfuge tubes each. Having paid for sequencing services online, label each tube correctly and send to the following address:

Eurofins Genomics,

Anzinger Str. 7a,

85560 Ebersberg,

Germany.

Upon receipt of the sequencing results, EXPASY is used to obtain the amino acid sequences, and thus to determine the success of site-directed mutagenesis.

2.24: Preparing overnight cultures in TBA

8 μL of amp is added to 8 mL of TB in a sterilin tube. 5 μL of IPTG is then added. The TBA is then inoculated with a looped colony of competent cells. Prepare another tube containing 8 mL of sterile TBA as a control. (It is good scientific practice to do all of this in the fume hood.) Store the tubes overnight at 37˚C in an incubator shaker. The following day, retrieve the tubes and examine their appearance. Successfully inoculated samples appear murky in colour. A cloudy appearance for TBA that has not been inoculated indicates contamination.

2.25: Preparing an SDS Polyacrylamide gel

Prepare two SDS-PAGE plates. Place a rubber gasket ridge-side-up around the furrowed plate. Lay the smooth plate on top of the latter, and secure them both with a clip on each side. Use distilled water to test if the plates are air-tight. Prepare a 15% resolving gel and a 4% stacking gel as follows:

                                                                   15% resolving gel        4% stacking gel

30% Acrylamide (mL)                                     3.75                              0.325

Distilled water (mL)                                        1.75875                       1.54

1.5M Tris-HCl pH 8.8 (mL)                             1.875                              –

0.5M Tris-HCl pH 6.8 (mL)                               –                                 0.625

10% APS (μL)                                                      37.5                             12.5

10% SDS (μL)                                                      75                                25

Add 3.5 μL of Temed to the resolving gel and pour it in. Add 0.5 – 1 mL of 20% IMS on top to resolve any bubbles, and wait approximately 40 minutes for the gel to set. At this point, add Temed to the stacking gel, pour out the IMS, and pour in the stacking gel. Slowly and gently slot in the comb, and wait roughly 30 minutes for the gel to set. If the gel is not to be used immediately, cover it with some paper towels, soak it with distilled water, and store it in a sealed bag at 4˚C.

2.26: SDS-PAGE, staining & de-staining

Prepare the SDS-PAGE box by connecting it to a power station and pouring in 1X SDS-PAGE buffer to the wire. Slot in the prepared gel at the front, with a flat plate at the back. Secure the plates, fill the box to the top with 1X buffer, and remove the combs. Run at 15 mA for 30 minutes. Prepare 1 in 5 dilutions (0.8 mL distilled water + 0.2 mL sample) and read the absorbance at 600 nm for each. Use this figure to calculate the volume of culture to be harvested as follows:

(0.7 / Absorbance X 5) X 300 = Volume of culture (μL)

Aliquot these volumes into new microfuge tubes, and top them all up to the highest value with distilled water. Centrifuge these samples at 13,300 rpm for 2 minutes. Discard the supernatant, and resuspend the pellet in 50 μL of 10X SDS buffer.* (Do this in the fumehood, while wearing gloves.) Heat the samples at 100˚C for 5 minutes.

Load 18 μL in each lane, as well as the ladder in at least one lane, and run the gel at 70 V for 10-15 minutes. A 2-212kDa protein ladder is used:

Picture5

Figure 5: Broad Range Protein Marker 2-212kDa visualised by Coomassie Blue staining on an SDS Polyacrylamide gel. © New England Biolabs Inc.

Once the samples have run out, ramp up the voltage to 120 V. Once the dye has run off – usually after 90 minutes – gently transfer the gel to an empty butter box. Stain the gel using Coomassie Blue staining solution.

To de-stain the gel, pour out the Coomassie Blue solution (reusable) and allow the gel to be washed in distilled water for 15 minutes. Discard the water and pour in Coomassie de-staining solution, leaving the gel on the shaker for the same amount of time it was left to stain. Pour out the de-staining solution and examine the gel.

* The procedure for protein fractions is the same, except that instead of pelleted cells being resuspended in 50 μL of 10X SDS buffer, 2 μL of 10X SDS buffer is added to each 18 μL of protein sample to be loaded.

2.27: Inducing protein expression

2 mL of the clone identified during screening is used to inoculate 200 mL of TBA. This culture is incubated in an incubator shaker at 37˚C, with the absorbance (600nm) read every hour. When this reaches 0.4 – 0.6, 200 μL of IPTG is added aseptically and the culture is transferred to a 30˚C incubator shaker for 16 hours.

2.28: Making up lysis buffer

NaH2PO4                      50 mM

NaCl                              0.5 M

Imidazole                     20 – 250 mM

pH                                  8.0

2.29: Stripping and recharging the IMAC resin

Note: 1 Column Volume (CV) = 2 mL.

The column is first washed with 2 CV of distilled water, followed by 2 CV of 50% ethanol. The metal ions are then stripped by washing with 2 CV of 100 mM EDTA, pH 8.0. To remove any remaining impurities, the column is then washed with 2 CV of 200 mM NaCl, 2 CV of distilled water and 10 CV of 30% isopropanol. The resin is then washed with 10 CV of distilled water and recharged with half a CV of 100 mM NiSO4. The column is again washed with 10 CV of distilled water and stored in 20% ethanol.

2.30: Cell lysis to obtain recombinant protein

Having induced expression with IPTG, the culture is retrieved and centrifuged at 5,000 rpm for 10 minutes at 4˚C using sterile centrifuge bottles. The supernatant is poured off and the pellet is resuspended in 100 mL of lysis buffer (20 mM imidazole) with 200 μL of anti-foam. The cells are lysed using a cell disruptor (Constant Systems Ltd.) at 15 kPSi twice. This cell lysate is collected in high speed centrifuge tubes and spun at 13,000 rpm for 40 minutes at 4˚C. The lysate is filtered through a Whatman filter into a clean duran.

2.31: IMAC

            Step                                           Added                                            Fraction Collected

  1. Rinse                             10 CV Distilled water                                         –
  2. Equilibrate                   10 CV Lysis buffer (20 mM)                              –
  3. Lysate                            Filtered lysate                                       Unbound lysate
  4. Wash                             10 CV Lysis buffer (20 mM)                20 mM wash X 100 mL
  5. Wash (alt.)                   10 CV Lysis buffer (80 mM)                 80 mM wash X 100 mL
  6. Elution                           5 CV Lysis buffer (500 mM)               12 X 1 mL
  7. Rinse                              10 CV Distilled water                                        –
  8. Storage                          In 20% IMS                                                          –

The protein fractions are visibly inspected using a transilluminator, to confirm their emission patterns.

2.32: Buffer exchange with PBS (protein purification)

Having inspected the protein elutions under UV light, the most concentrated fractions are buffer exchanged into 1X PBS for further spectral and visual analysis. Do this as follows:

Aliquot 500 μL of protein elution into a Vivaspin column, and centrifuge at 13,000 rpm for 10 minutes. Repeat this until all of the protein has collected in the filter membrane (10,000 MWCO). Wash the column once with PBS. Resuspend the collected protein in PBS and measure its absorbance at 280nm. (1X PBS is used as a blank.)


Chapter 3: Diary of Work Done, Results & Data Interpretation

3.1: Prelude

In writing this report, the author opted to use a diary-like format: that is, a consecutive flow of the physical work done, the results of this work, and discussion of the results. There are two reasons for this:

1. A clear statement of the work done each day is the best evidence of having kept a proper diary during the project (bar handing up the rough copy, which is in no condition to correct). This is a necessary skill for any scientist.

2. By having each set of results immediately followed by the discussion, there is no need for the reader to flip back and forth between separate sections of the report.

A meeting was scheduled with Dr. Brendan O’Connor prior to the commencement of the project. This enabled the author to draw up a schematic of the work ahead, which was as follows:

Picture6

Figure 6: Flow diagram of the physical work involved.

The author was also encouraged to have an attempt at primer design. This was successfully carried out by drawing upon the knowledge gained in 3rd year microbiology practicals, including how:

  • The primers need to be approximately 30 base-pairs in length, with a high G-C content (more than 65%) at the 3’ end;
  • The sequence of the reverse primer should be complementary to the 30 bp immediately preceding the forward primer;
  • The primers should be designed such that the complementary region has a Tm of around 60˚C;
  • PCR primers usually come without a phosphate group on their 5′ termini. As a result, we cannot simply ligate the ends of a PCR product together; rather, they must be phosphorylated first. This can be circumvented by ordering the primers with a phosphate already added to the 5′ end.

3.2: Diary

Day 1

Preparation of agar plates –– Cultures of pQE-30 (carrying DsRed) in LBA –– Cultures of KRX and JM109 in LB

Our first task was to make up 1500 mL of LB broth (Section 2.1). To prepare for the first round of transformation on Day 7, 1200 mL of this broth was used to make up LBA agar plates (Section 2.3); the remaining 300 mL was divided into two 150 mL stocks. Ampicillin was added to one of these stocks (Section 2.2) for use in culturing our pQE-30 plasmid carrying DsRed (Section 2.5); the remaining amp-free LB was used to culture KRX and JM109 – our competent cells – to test their confluency (Section 2.4).

The vector pQE-30 (PROMEGA) flourishes in LBA, as it carries an AmpR marker. It expresses DsRed such that the protein is bound to a poly-Histidine tag, thereby enabling its purification by immobilised nickel affinity chromatography.

KRX and JM109 (PROMEGA) are high expression E. coli strains, designed to optimise transformation and the induction of protein expression. (More details are provided on Day 6.)

Day 2

pQE-30 confirmation gel –– Preparation of TE Buffer –– Stocks of pQE-30 in LBA –– Stocks of KRX and JM109 in LB

Our overnight cultures of pQE-30 in LBA were a success. The plasmid was then purified from the medium (Section 2.6) and run through agarose gel electrophoresis (Section 2.7), to confirm its identity on the basis of size (3641bp). The success of this gel would enable us to proceed with mutagenesis PCR on Day 3: the induction of V105A. (The bands representing pQE-30 in lanes 2 and 3 were rather faint, indicating a low cell number; this can be put down to inadequacy in our DNA purification technique.)

TE Buffer was also made up (Section 2.8) to prepare for PCR on Day 3. Resuspending the primers in TE Buffer solubilises them, while protecting them from degradation. (Tris inactivates DNA nucleases by adjusting the pH to 8.0; EDTA does so by binding to metal cations that are required by the nucleases.)

Stocks of the pQE-30 cultures in LBA were prepared as well (Section 2.9). This would enable us to grow up the plasmid, induce the expression of DsRed, purify the protein, and compare it with our final mutant at the end of the project.

Our overnight cultures of KRX and JM109 in LB were also successful, thus demonstrating their confluency. Stocks of both strains were prepared as a precautionary measure (Section 2.9), i.e. in case problems were to arise with the competent cells to be prepared on Day 5.

Day 3

Resuspension of primers in TE Buffer –– Site-directed mutagenesis, round 1/3

Before proceeding with PCR, it was necessary to resuspend the primers in TE Buffer and to make 1 in 10 dilutions of these stocks (Section 2.10). The first round of mutagenesis PCR – changing DsRed to E5up – was then carried out, with four aliquots of 50 μL for the four potential annealing temperatures: 55, 60, 65, and 70˚C (Section 2.11). On Day 4, an agarose gel would be run to confirm the success of our PCR.

Day 4

PCR confirmation gel –– Repeat of PCR –– PCR confirmation gel –– DNA purification from PCR

The PCR samples were run on a 0.7% agarose gel to confirm the presence of our newly synthesised plasmid, presumably now carrying E5up. The results of this gel were poor: there was nothing in any of the lanes representing our plasmid. It is highly likely that 1 μL of template DNA was added to the PCR master mix instead of 4 μL. Thus, there was a need to repeat the PCR. The results of the second confirmation gel were as follows:

Picture7

Figure 7: Results of PCR confirmation gel for pQE-30, now carrying E5up.

The plasmid is linearised; therefore, it should be more than 4000 bp in size. This is represented in lane 4 (55˚C annealing temperature). The plasmid in lane 5 is not the correct size – the result of primers binding to a different sequence. When lane 6 was loaded, the bottom of the well was pierced, causing the plasmid to leak out. The bands in lane 7 are smeared, suggesting DNA degradation. As for lanes 1 and 2, these represent unsuccessful attempts at loading the marker ladder.

Upon confirmation, the plasmid was purified from the PCR mix (Section 2.12) to prepare for a restriction digest on Day 5. The purpose of the restriction digest is to remove the template DNA and leave us with the new mutant sequence.

Day 5

Restriction digest –– Preparation of TB and SOB –– Cultures of KRX and JM109 in LB –– Preparation of SOC

A restriction digest was carried out to cut away the template DNA from our purified plasmid, now carrying E5up (Section 2.13).

Terrific Broth (TB) and Super Optimal Broth (SOB) were also prepared for use in growing up our competent cells on Day 6 (Sections 2.14 and 2.15, respectively). To this end, overnight cultures of KRX and JM109 in LB were prepared as well.

The remaining SOB was used to make up SOC (SOB with catabolite repression, Section 2.16), to be used in the transformation of competent cells on Day 7.

Day 6

SyBr Safe gel –– DNA purification from gel –– Ligation –– Growing up KRX and JM109 competent cells

The nicked plasmids were run on a SyBr Safe gel (Section 2.17) to confirm the success of our restriction digest and to excise the best expressed plasmid. We expected to see multiple bands representing the removed template DNA, and one ~4000bp band for the plasmid. However, this was not the case: rather, there was nothing on the first SyBr Safe gel. Given the success of the PCR, we had to conclude that mistakes were made in the restriction digest (e.g. the buffer not being mixed properly), causing the plasmid to leak out of the wells.

A faint band for our plasmid was achieved on the second attempt. We excised this band from the gel, purified the cut plasmid (Section 2.18), and sealed it up by carrying out a ligation (Section 2.19). The plasmid was now ready to be taken up and expressed by our competent cells. (If an improperly ligated plasmid is taken up and expressed by bacterial cells, the sequence is more prone to random mutation.)

The second requirement for transformation was to grow up the KRX and JM109 competent cells, using our LB cultures (Section 2.20). During the incubation step, it is important to monitor the absorbance levels of the cells, as an indicator of concentration. The optimum absorbance is 0.4 – 0.6 (read at 600nm).

The JM109 strain is more suited to transformation than KRX. Since JM109 lacks the E. coli K restriction system, recombination of cloned DNA with host chromosomal DNA is prevented. (The endonuclease A-mutation also results in an improved quality and yield of isolated plasmid DNA.) To illustrate this point, both the JM109 and the KRX aliquots would be used in the first round of transformation on Day 7.

However, since KRX is more suitable for induced protein expression (owing to the presence of a chromosomal copy of T7 RNA polymerase under the control of rhaBAD, a ramnose promoter, allowing for dramatic control over the proteins that can be expressed prior to induction), after round 3 of PCR, only the KRX transformants would be used for the induced expression and purification of our mutant protein.

Day 7

Transformation of KRX and JM109 with pQE-30 carrying E5up

Our KRX and JM109 competent cells were transformed with the ligated plasmid (Section 2.21), thus enabling (and optimising) the expression of E5up – our enhanced DsRed protein. We would check for single, fluorescent colonies on Day 8. These colonies would be used to make overnight cultures in LBA, in preparation for the second round of PCR: the induction of S197T to change E5up to E5.

When we heat-shock KRX and JM109 to take up the plasmid, they gain AmpR. This is what enables them to survive on the LBA agar plates.

Day 8

Cultures of JM109 expressing E5up in LBA

Single colonies were present for the JM109 plates, but not the KRX plates. While we certainly expected a higher rate of transformation for JM109 than KRX, the complete absence of KRX transformants was surprising. Given that there were only three colonies present on the JM109 plate, this can be attributed – at least in part – to our inexperience at transformation.

Picture8

Figure 8: Results for transformation of concentrated JM109 with pQE-30 carrying E5up (enhanced red), viewed under the transilluminator.

Figure 8 above displays the three highly fluorescent single colonies obtained on our concentrated JM109 plate. Numerous less fluorescent colonies are also visible. In the case of the latter, it is possible that E5up is being expressed by the cells, but the protein has yet to lose most of its residual green fluorescence, thus compromising the enhanced red. It is also possible that these cells have grown on patches of agar with no amp, and are now dying – the result of not mixing the amp sufficiently with the agar.

Figure 9 below sheds more light on the situation, and also serves as a warner against loading two different fluorescent proteins on the same SyBr Safe gel:

Picture9

Figure 9: Contamination of GFP with DsRed (expressed by JM109 and/or KRX).

Colonies of JM109 and/or KRX expressing E5up are visible among the mass of yellow-green colonies (presumably expressing EYFP) belonging to colleague Gary Gillick. Since we shared the same SyBr Safe gel, it is safe to conclude that leakage from our wells settled in his. Undoubtedly, this loss of plasmid concentration played a role in the scarcity of fluorescent single colonies on our plates. To guard against cross-contamination in the future, we would make sure to run our digests on separate gels.

Proceeding with the assumption that we had obtained single colonies of JM109 expressing E5up, overnight cultures were prepared in LBA. The plasmid would be purified on Day 9, to prepare for round two of PCR.

Day 9

DNA purification from LBA –– Site-directed mutagenesis, round 2/3 –– Stocks of JM109 expressing E5up in LBA

Under the transilluminator, our overnight cultures glowed bright red; thus, we had achieved 5 mL cultures of JM109 expressing E5up. The plasmid was subsequently purified and used for the second round of PCR: the induction of S197T to change E5up to E5.

As was done for DsRed, stocks of JM109 expressing E5up were prepared for comparing our mutants at the end of the project.

Day 10

PCR confirmation gel

The plasmids were run a 0.7% agarose gel, to confirm the success of our PCR. The results were as follows:

Picture10

Figure 10: Results of PCR confirmation gel for pQE-30, presumed to be carrying E5.

A band of approximately 4000bp can be seen in lane 5 (70˚C annealing temperature). This was the only band on the gel; the smear in lane 2 (representing millions of copies of plasmid DNA) tells us that the 55˚C sample was degraded.

We wanted the maximum amount of E5 to work with, so we needed to be reasonably certain that only the 70˚C sample contained the newly synthesised plasmid. To this end, a 40 minute gel was run with double the number of samples. The results of this gel were as follows:

Picture11

Figure 11: Results of 40-minute PCR confirmation gel for pQE-30, with twice the number of samples.

Once again, the only bands that appeared were for the 70˚C sample (lanes 5 and 10). Thus, we would proceed with DNA purification from PCR tube 4 on Day 11.

Days 11 & 12

DNA purification from PCR mixture –– Restriction digest –– SyBr Safe gel –– DNA purification from gel –– Ligation –– Preparation of agarose gels

These two days were spent preparing for the second round of transformation. Upon purification of pQE-30 – now carrying E5, presumably – from the PCR mix, a restriction digest was carried out. The resulting digests were run on a SyBr Safe gel, with the best expressed plasmid being excised, purified and ligated. The plasmid was now ready to be taken up and expressed by our competent cells on Day 13.

There was a worry that the elution buffer in the miniprep kit was at fault. Thus, it was decided that distilled water would be used from now on.

Three agarose gels were also made up for future use. These gels were stored at 4˚C with a small amount of 1X TAE buffer, to keep them from drying out.

Day 13

Transformation of JM109 with pQE-30 carrying E5 –– Preparation of SOC

Our JM109 competent cells were transformed with the ligated plasmid, to achieve the optimal expression of E5. For the reasons given on Day 6, only the JM109 aliquots were used this time; the KRX aliquots would be used for the third round of transformation and subsequent protein expression analysis.

For the concentrated plates, given the large bands obtained for our plasmid on Day 10 (second PCR gel), it was decided to prepare a 1 in 4 dilution, to ensure the presence of single, fluorescent colonies on Day 14.

It was necessary to make up SOC (from 100 mL of SOB) before proceeding with the transformation, as the previous stock had become contaminated. SOC contains more nutrients than LB, and thus has a shorter shelf life.

Just before proceeding with the spread plates, however, it was discovered that all of our LBA agar plates had been contaminated with fungus. Given that the plates were prepared under sterile conditions and kept at 4˚C, it is likely that the agar simply wasn’t allowed to cool down sufficiently when amp was first added, thus negating its effects and allowing for eventual contamination from a variety of sources.

Donal Moynihan, a PhD student at the ISSC, permitted us to use some of his plates instead. We would return the favour on Day 14 by making up fresh plates.

Day 14

Preparation of SOC –– Preparation of agar plates

Not one colony was present on anyone’s plates. While it is common knowledge that rates of success for transformation decrease after each round, once again, the complete lack of transformants was surprising. Probability dictates that something all three of us were working with was at fault, rather than all three of us making serious errors in the transformation. This may well have been the SOC broth, as it was not mixed properly in its preparation (the heat-stir wasn’t used). This was made up again to repeat the transformation on Day 15.

20 fresh plates of LBA agar were also prepared, to replace the ones given to us on Day 13.

Day 15

Transformation of JM109 with pQE-30 carrying E5 –– Streak plates of JM109 expressing E5up

We repeated the transformation of our JM109 competent cells with pQE-30, presumed to be carrying E5.

In addition, the one fluorescent colony of JM109 expressing E5up obtained on our neat plate (Day 8) was used to streak for single colonies (Section 2.22). On Day 16, these colonies would be used to make overnight cultures in LBA, from which we would purify our plasmid to send for sequencing. We needed to be absolutely certain that the first mutation was induced; for if it was not, then proceeding to induce the other mutations required to obtain AG4 would be pointless.

Day 16

Transformation of JM109 with pQE-30 carrying E5up –– Culturing pQE-30 carrying E5up in LBA

Once again, there were no colonies on the plates for any of us. To narrow down the possibilities of what went wrong, it was decided to repeat the first transformation, i.e. the uptake and expression of pQE-30 (carrying E5up) by KRX and JM109. We reasoned that if this worked – and there was no reason to think that it wouldn’t, as it worked the first time – then there were three potential issues:

1. Something went wrong in preparing E5 for transformation – possibly the ligase becoming faulty;

2. The plates were at fault (unlikely, as they were prepared by the book);

3. The SOC was at fault (unlikely, as extra care was taken in its preparation).

There was a need to make up more LBA agar plates before proceeding with the transformation. It was also decided that LB broth would be used instead of SOC from now on, as it is less prone to contamination. We would check for single colonies on Day 17.

On our E5up streak plate, there was an abundance of fluorescent single colonies. These were used to inoculate overnight cultures of LBA. This would be followed up with a plasmid prep on Day 17.

Day 17

Ligation –– DNA purification from LBA

Yet again, nothing appeared on our plates. This was surprising, as our previous E5up transformation worked just fine; clearly there was nothing wrong with our ligated plasmid. Neither should there have been anything wrong with our competent cells, as they had been kept at -80˚C the whole time. The ligase becoming faulty may explain the failure of our E5 transformation attempt, but it doesn’t explain this; clearly there was nothing wrong with the ligase originally. There was no issue with our plates either (no contamination, agar prepared in separate 500 mL jars for each of us) or our LB, for the same reasons.

At the time, we simply had to be content with this gap in our understanding. All we could do was proceed with the assumption that our plasmid (carrying E5) wasn’t ligated properly. There was just enough of the purified E5 digest left over; thus, we performed a ligation (with a fresh tube of ligase, to rule out the possibility of it being an issue next time) and would attempt transformation again on Day 18.

We also purified pQE-30 (carrying E5up) from the cultures in LBA. The purified ligation would be used for PCR on Day 18, to provide us with E5 in case the new transformation doesn’t work. We would also send the ligation for sequencing on Day 19.

Day 18

Transformation of JM109 with pQE-30 carrying E5 –– Site-directed mutagenesis, round 2/3 (repeat)

Using our fresh E5 ligation, the transformation of JM109 was attempted once more.

By this time, we had concluded that since the failure of our second E5up transformation had nothing to do with the ligase, clearly there was some issue with our competent cells. Perhaps the aliquots used for the second E5up transformation were faulty, whereas those used for the first E5up transformation were just fine.

We reasoned that with our newly ligated plasmid (carrying E5), if the transformation still doesn’t work, then it would be safe to conclude that our competent cells have been compromised. In this case, we would use Donal’s competent cells for transformation on Day 20, using the plasmid to be obtained from PCR on Day 19 (carrying E5).

On the other hand, if the transformation does work, then at least some of our competent cell stocks are fine. In this case, we would make overnight cultures on Day 19 and proceed with the third round of PCR (inducing V71M to obtain AG4) on Day 20.

The second round of PCR was repeated for some of our first ligation (carrying E5up), to provide us with a backup of E5. The remainder would be sent for sequencing on Day 19.

Day 19

PCR confirmation gel –– DNA purification from PCR mixture –– Restriction digest –– SyBr Safe gel –– DNA purification from gel –– Ligation –– Sending plasmid DNA for sequencing

The transformation didn’t work, thus demonstrating that our JM109 cells were no longer competent. Having been provided with fresh competent cells by Donal, we prepared our newly synthesised plasmid (carrying E5) for what would hopefully be the final attempt at transformation on Day 20.

The plasmids were first run on an agarose gel, to confirm the success of our PCR. The results were as follows:

Picture12

Figure 12: Results of PCR confirmation gel for pQE-30, presumably now carrying E5.

Bands of around 4000bp could be seen for the 65 and 70˚C samples, thus indicating the presence of E5. The double band obtained in lane 4 suggests the presence of a large contaminant with our plasmid; thus, we only combined the 65 and 70˚C samples.

After this, we purified the plasmid from the PCR mix, performed a restriction digest, and ran the digests on a SyBr Safe gel. The most concentrated band was excised, and the DNA was purified and sealed up with a ligation.

Our purified ligation from Day 18 (carrying E5up) was sent for sequencing (Section 2.23).

Day 20

Transformation of JM109 with pQE-30 carrying E5

The new aliquots of JM109 were transformed with our freshly ligated plasmid, carrying E5. We reasoned that if this worked, then we would know with absolute certainty that our competent cells were the problem.

Day 21

Streak plates of JM109 expressing E5

At long last, fluorescent single colonies appeared for our E5 transformation. We could thus say with certainty that the competent cells were at fault. Perhaps they were allowed to thaw out for too long in our first attempt.

Picture13

Figure 13: Results for transformation of JM109 with pQE-30 carrying E5 (enhanced red), viewed under the transilluminator.

Five of these colonies were used to prepare streak plates, to supply us with an abundance of colonies for making overnight cultures on Day 23.

Day 22

Cultures of JM109 expressing E5 in LBA

Five LBA samples were inoculated with single colonies of JM109 expressing E5. On Day 23, we would purify our plasmid from the best expressed clone, and proceed with round three of PCR: the induction of V71M to obtain green-emitting AG4.

Day 23

Stocks of JM109 expressing E5 –– DNA purification from LBA –– Site-directed mutagenesis, round 3/3 –– PCR confirmation gel –– Sequencing results for E5up

Our LBA samples were successfully inoculated. As was done for DsRed and E5up, stocks of JM109 expressing E5 were prepared for comparing our mutants at the end of the project. The plasmid was then purified from the LBA, and used for the third round of PCR. Once the PCR had run its course, the newly synthesised plasmid (presumably now carrying AG4) was run on a 0.7% agarose gel.

According to the results (no image available), annealing and extension occurred for our 60, 65 and 70˚C samples. As before, however, there was a concern that the band for our 65˚C sample might consist of two plasmids; therefore, only the 60 and 70˚C samples were combined. We would proceed with DNA purification on Day 24.

As a side note, here is what happens when DNA samples are run on an agarose gel that hasn’t set properly:

Picture14

Figure 14: The results of agarose gel electrophoresis when the agarose hasn’t set properly.

Although the bands are all of the same size, the incomplete solidification of the gel has affected their positions. (This happened to us at least once.) It is important to take such basic considerations into account when faced with data that doesn’t appear to make sense.

~

The sequencing results had finally arrived for E5up. The data gave us the same exact sequence as DsRed, but with one crucial difference: at amino acid 105, there is an Alanine instead a Valine. This is highlighted in blue below:

Picture15

Figure 15: The amino acid sequence of DsRed-E5up (EXPASY translation, forward primer) showing the replacement of Valine with Alanine at position 105.

Our primers were designed to incorporate a Cysteine instead of a Thymidine at nucleotide 314, to alter GTG to GCG; this results in Alanine instead of Valine. Thus, we had successfully induced the first of three mutations required to obtain AG4.

Day 24

DNA purification from PCR mixture –– Restriction digest –– SyBr Safe gel –– DNA purification –– Ligation

Our newly synthesised plasmid – presumed to be carrying AG4 – was purified from the PCR mix. This was followed by a restriction digest, and the digests run on a SyBr Safe gel. The most concentrated band was excised, and the nicked plasmid was purified and ligated. This left us in a prime position for transformation on Day 25.

Day 25

Transformation of JM109 with pQE-30 carrying AG4

The JM109 cells provided to us by Donal were transformed with our newly ligated plasmid. We reasoned that if green fluorescent colonies appeared on our plates, then we could be reasonably sure that V71M was successfully induced.

Day 26

Streak plates for red and non-fluorescent colonies of JM109 –– Transformation of JM109 with pQE-30 carrying AG4 –– Ligation

There were a few red fluorescent colonies on the concentrated plate, as well as an abundance of colonies displaying no fluorescence (no image available). We reasoned that either the mutation worked, or it did not. If the mutation did not work:

1. Red colonies are E5 (or E5up), non-fluorescent colonies are faulty E5 (or E5up);

2. Red colonies are contamination from E57, non-fluorescent colonies are faulty E5 (or E5up).

The induction of S197T does not alter the red fluorescence, so there is no visible difference between E5up and E5. Conversely, if the mutation worked:

1. Non-fluorescent colonies are AG4, red colonies are contamination from E57;

2. Non-fluorescent colonies are AG4, red colonies are contamination from E5 (or E5up).

Knowing that the induction of V71M negates the enhanced red fluorescence, and assuming that we had induced S197T, we speculated that perhaps the green fluorescence is only visible under UV light; we would check this after purifying the protein by IMAC. Sequencing would confirm the protein’s identity long before this.

Streak plates were prepared for both the red and non-fluorescent colonies, thus enabling the preparation of overnight cultures on Day 27.

For the purposes of protein expression analysis, the AG4 transformation was also repeated using KRX. LBA and TBA were made up to this end.

Our purified ligation (carrying E5) was also sent for sequencing.

Day 27

Cultures of red and non-fluorescent colonies of JM109 in LBA and TBA –– Preparation of SDS Polyacrylamide gel –– Streak plates of KRX colonies

Cultures of both the red and non-fluorescent colonies obtained for our Day 26 streak plates were prepared in LBA and TBA (Section 2.24). On Day 28, the TBA cultures would be used to identify our best expressed clone via SDS-PAGE. The LBA cultures would then be used for the induced expression and purification of this clone. As explained on Day 6, the latter would only be done for KRX; this would make the JM109 gel a practice run, so to speak.

An SDS Polyacrylamide gel was also prepared for this purpose (Section 2.25), and stored overnight at 4˚C. (Heating the samples in SDS buffer coats the proteins in a negative charge. As a result, when the gel is run, all the proteins move towards the positively-charged electrode, meaning they separate on the basis of size, rather than charge. This enables the identification of any potential contaminants.)

Streak plates were also prepared using the colonies obtained for our KRX transformation. The resulting single colonies would be used to make overnight cultures on Day 29.

Day 28

Stocks of M3 Red and M3 Green in LBA –– SDS-PAGE for M3 Red and M3 Green

The cultures of non-fluorescent colonies prepared in LBA and TBA were now fluorescing yellowish-green; we presumed this was AG4. The cultures of red colonies were now super-red; we presumed this to be E5 (or E5up), or possibly contamination from colleague Andrius’ E57 plasmid (also enhanced red). From now on, these proteins would be referred to as M3 Green and M3 Red respectively.

Our cultures for M3 Red and M3 Green (JM109) in LBA appeared as follows:

Picture16

Clearly we had a different fluorescent protein for M3 Green, though perhaps not AG4; the sequencing results would reveal all. Stocks of both were prepared for this purpose.

The difference between our overnight cultures in TBA was less striking. However, by centrifuging 1 mL of each to pellet the cells, it was possible to confirm their different fluorescence patterns.

Therefore, we proceeded to use both of these cultures for SDS-PAGE (Section 2.26), with the gel to be de-stained on Day 29. Ideally, this would enable the identification of our best expressed clone, as would be done for KRX on Day 31.

However, the gel didn’t run properly, as the gaskets were left on: rubber insulates against electricity. Thus, the gaskets were removed, more SDS-PAGE buffer was added, and the gel was rerun. It would become clear how this affected the protein on Day 29.

Day 29

DNA Purification from glycerol stocks (error) –– Sending M3 Red and M3 Green for sequencing –– Streak plates for KRX single colonies

Unsurprisingly, the de-stained JM109 gel appeared distorted:

Picture17

Figure 17: Distorted SDS Polyacrylamide gel. Proteins do not separate out well unless they are forced to migrate by an opposing electrical charge. The rubber gasket, if left on, acts as a barrier between the proteins and this charge.

Bands were present for our proteins, but it was impossible to tell whether or not they were the correct size (~28 kDa). Thus, we would exercise particular caution in readying our KRX gel on Day 31.

Using the stocks made on Day 28, our ligations carrying M3 Red and M3 Green were purified and sent for sequencing. This was a mistake, as the cells need to cultured in LBA first: up to a third of the cells stored at –80˚C in glycerol die straight away. Thus, we expected the sequencing results to be inconclusive.

As regards the KRX streak plates prepared on Day 27, although single colonies were present, there was not an abundance of them; this was due to poor streaking technique. Thus, five more plates were streaked in the correct manner, with the resulting colonies to be used for the preparation of overnight cultures on Day 30.

Day 30

Cultures of KRX expressing M3 Red and M3 Green in LBA and TBA –– Preparation of SDS Polyacrylamide gel

Using the colonies obtained on our new KRX streak plates, overnight cultures of KRX expressing M3 Red and M3 Green were prepared in LBA and TBA, for protein screening on Day 31.

An SDS Polyacrylamide gel was also prepared for this purpose, to be stored overnight at 4˚C.

Day 31

SDS-PAGE for M3 Red and M3 Green –– Cultures of KRX expressing M3 Red and M3 Green in LBA and TBA

The TBA was successfully inoculated; thus, we proceeded with SDS-PAGE for KRX expressing M3 Red and M3 Green.

The cultures in LBA, on the other hand, were contaminated. Thus, fresh LBA was made up for a redo of our overnight cultures. Following the identification of our best expressed clone, these cultures would be used to inoculate 200 mL of TBA, for the induction of protein expression.

Day 32

Cultures of KRX expressing M3 Red and M3 Green in LBA and TBA –– Preparation of SDS Polyacrylamide gel

On the de-stained KRX gel, the correct-sized bands were visible for M3 Red (lanes 3-5), with our best expressed clone in lane 3. However, there were no definitive bands for M3 Green (lanes 6-7). The bands that were present in lanes 6-7 were not the correct-size (~28 kDa), thus, we could not proceed to induce the expression of M3 Green. (No image available.) We reasoned that it was possibly a partially-ligated plasmid, and thus was not expressing as it should have been; or perhaps poor loading technique was the source of the problem.

At this point, the sequencing results had yet to arrive for either plasmid; thus, on Day 33, SDS-PAGE would be repeated for both M3 Green and M3 Red, for the sake of consistency. This necessitated making up new overnight cultures in TBA and LBA, as well as a new SDS Polyacrylamide gel.

Day 33

SDS-PAGE for M3 Red and M3 Green –– Stocks of KRX expressing M3 Red and M3 Green in LBA

Using the overnight cultures prepared in TBA, KRX was rerun through SDS-PAGE for M3 Red and M3 Green. The gel would be de-stained on Day 34.

Assuming that the correct-sized bands (~28 kDa) would be present for both proteins, stocks were made from our cultures of KRX in LBA.

Day 34

Cultures of KRX expressing M3 Green and M3 Red in LBA

The de-stained gel for KRX was identical to the previous one: sizeable bands appeared for M3 Red (some bulkier than others), while no definitive bands appeared for M3 Green.

Just in case the bands for M3 Green were not telling the whole story, on Day 35, we would induce the expression of both proteins. Before doing so, however, there was a need to make up new cultures in LBA, using the stocks prepared on Day 33.

A note on induced protein expression

Along with obtaining a high concentration of protein, one reason that we always try to keep the environment induced is the susceptibility of Methionine residues to oxidation – a problem when we have very pure solutions of protein. As the Methionine becomes modified, the result is a misfolded protein response. This oxidation occurs often, and is a key problem in large-scale protein purification. In fact, there is a large focus on engineering out this Methionine in protein drugs, as modified Methionines can sometimes lead to an antigenic protein.

Day 35

Preparation of TBA –– Stripping and recharging the IMAC resin –– Inducing the expression of M3 Red and M3 Green –– Preparation of lysis buffer

Using the KRX cultures prepared in LBA on Day 34, we proceeded to induce the expression of M3 Red and M3 Green, using IPTG (Section 2.27).

There was a need to make up 400 mL of TBA before proceeding. There was also a need to strip the IMAC resin of any nickel ions to which proteins may be bound, and recharge it with 100mM NiSO4 (Section 2.29).

IPTG is an acronym for Isopropyl β-D-1-thiogalactopyranoside, a synthetic analogue of lactose that is highly stable. IPTG inactivates the lac repressor in E. coli and promotes lactose utilisation by inducing the synthesis of beta-galactosidase. IPTG is thus used to induce the expression of cloned genes that are under control of the lac operon in E. coli.

After the addition of IPTG, it takes approximately two hours for the KRX cells to reach an absorbance of 0.4 – 0.6. After 2.5 hours, our KRX cells expressing M3 Red had an absorbance of 0.562. However, the cells expressing M3 Green only had an absorbance of 0.093, which absolutely does not indicate cell growth. We reasoned that our KRX cells expressing M3 Green had become contaminated: after all, the aliquots used for absorbance measurement were not taken in the fume-hood, as they should have been. This being the case, we would postpone obtaining M3 Green protein until Day 44, and continued with the expression and purification of M3 Red.

On Day 36, we would obtain M3 Red protein using a cell disruptor, and then purify it via IMAC. To prepare for the latter, various concentrations of Lysis buffer were made up (20, 80 and 250mM imidazole). (See Section 2.28.)

Day 36

Lysis of KRX cells to release M3 Red protein –– Protein purification by IMAC –– Absorbance

On Day 35, once our induced KRX cells had reached the optimum absorbance, the LBA was left to incubate at 30˚C for 16 hours. On this day, the cells were lysed to obtain M3 Red (Section 2.30), which we proceeded to purify by IMAC (Section 2.31). The filtered lysate, the unbound lysate, the various washes, and the protein elutions were all collected. On Day 37, we would be able to assess the purity of our fractions via SDS-PAGE.

Our most concentrated M3 Red elution appeared as follows under the transilluminator:

Picture18

Figure 18: M3 Red protein (presumed to be E5up, E5, or E57) in lysis buffer (500 mM imidazole) after purification by immobilised nickel affinity chromatography. Evidently, the maturation of the DsRed fluorophore has reached its conclusion by the induction of V105A.

However, the brightness of our elutions was no guarantee that they were free of any major contaminants. This would become clear upon reading the absorbance on Day 38.

A note on IMAC

Our affinity column contains a Nitrilotriacetic acid resin, charged with Ni2+. Once the filtered lysate for M3 Red is added, the protein’s high affinity for Ni-NTA allows for the poly-Histidine tag to bind to the immobilised nickel ions on our resin. Most of the contaminant proteins in the added sample are washed through the column; those that bind have a weaker affinity for the resin, and are thus washed away using 20, 50, 80 and 100mM concentrations of imidazole buffer. M3 Red is then eluted by simply using a higher concentration of imidazole (200-250mM), as this causes the displacement of the His-tag.

As for the tag itself, this consists of six Histidine residues in tandem, with an enterokinase cleavage site to allow its ultimate removal. Six seems to give us the optimal affinity binding to immobilised nickel. Histidines carry negative charges at neutral pH levels (7.0-7.4); thus, with six Histidines in tandem, we have a poly-anionic tag that has a very high affinity for a divalent cation like nickel – higher even than that of antibody for antigen, or enzyme for substrate. Binding of the His-tag does not depend on the 3-D structure of the protein; rather, even if the tag is inaccessible, it will bind so long as two or more Histidines are available to interact with the nickel cation.

Day 37

SDS-PAGE for M3 Red

Our M3 Red fractions were run through SDS-PAGE, to investigate the presence of impurities. Any residual cell debris should have eluted in the unbound lysate; any major contaminant proteins should have eluted in the 20 mM and 80 mM imidazole washes. The gel would be de-stained on Day 38, thus giving us our answer.

Day 38

Buffer exchanging M3 Red in PBS –– Reading absorbance –– Fluorescence under UV light

The de-stained gel provided us with the ideal results:

1. Most of the contaminant proteins were eluted in the imidazole washes, though some remained in the M3 Red elutions.

2. The 28 kDa band obtained for our protein elutions (lanes 3-9) was present for the filtered lysate (lane 10, circled in red), but was absent for the unbound lysate (lane 11) and imidazole washes (lanes 12 and 13). This indicates that virtually all of the protein ended up binding to the column, remained bound during the washing steps, and was only eluted with the addition of 500 mM imidazole lysis buffer.

Picture19

Figure 19: The results of SDS-PAGE for our M3 Red elutions, the filtered lysate, the unbound lysate, and the imidazole washes.

The enduring presence of contaminants necessitated buffer exchanging our fractions into 1X PBS (Section 2.32); this was done for our most concentrated samples. Any contaminant proteins were expected to be resolved here. However, according to the absorbance readings, this was not to be; rather, a second large peak appeared on the spectrophotometer:

Picture20

Figure 20: Absorbance readings for our buffer-exchanged M3 Red elutions. The presence of another tall peak at ~650nm suggests that a large unidentified protein has remained in the elution.

This indicated the presence of another large protein in our samples, a protein that somehow eluded the washing steps. (This was not an imidazole peak, as PBS would have washed it out.) We posited that this was a subunit of our protein, but it was difficult to know without any sequencing results at hand. Without them, there was no option but to repeat the induced expression of M3 Red, and purify it again via IMAC. A gradient imidazole wash would be used this time, to completely remove any and all contaminant proteins.

On Day 39, we would prepare overnight cultures of M3 Red and DsRed in LBA, using the stocks in our possession. Prior to this, we were to run our buffer-exchanged fractions through SDS-PAGE to confirm the presence of this suspected contaminant protein and, if possible, identify it. (The purpose of growing up and purifying DsRed is to be able to demonstrate a shift in absorbance from our original protein to our mutant, representing the enhanced fluorescence.)

Our samples were also viewed under UV light. Our M3 Red elution exhibited a considerably more pink fluorescence than what was presumed to be E57 (enhanced red):

Picture21

Figure 21: Our M3 Red elution (left) compared to presumed E57 (right) under UV light. The sequencing results would confirm if this amounted to an actual difference in nucleotides.

Day 39

SDS-PAGE of buffer-exchanged M3 Red –– Cultures of KRX expressing M3 Red & JM109 expressing DsRed in LBA –– Sequencing results for M3 Red and M3 Green –– Cultures of KRX expressing M3 Green in LBA

Our buffer exchanged M3 Red fractions were run through SDS-PAGE. De-staining the gel on Day 40 would allow us to identify the contaminant protein responsible for the second absorbance peak. Given the concentration of our sample, dilutions to 1 in 6 were prepared and run on the gel alongside the neat protein.

As the gel was running, however, the power station kept switching between volts and amps. This was only noticed approximately 45 minutes after the beginning of electrophoresis, so this may have been the case from the start. There was a concern that this stop-start behaviour could cause the fractions to be smeared or appear at different positions on the gel. This would make it difficult to identify the contaminant protein; we would find out on Day 40.

Assuming the presence of this contaminant protein, on Day 40, we would repeat the induced expression of M3 Red, and also induce the expression of DsRed. To prepare for this, overnight cultures of KRX (expressing M3 Red) and JM109 (expressing DsRed) were prepared in LBA.

The sequencing results had finally arrived for M3 Red and M3 Green (nothing yet for E5). However, as expected, the results were inconclusive: we only received half of the sequence for M3 Red, and nothing for M3 Green (“ERROR”). Undoubtedly, this is because the plasmids were not grown up in LBA before being purified and sent off: rather, they were merely purified from the glycerol stocks. The plasmids need to be given a chance to be expressed, as explained on Day 29.

In accordance with this, an overnight culture of KRX expressing M3 Green was prepared in LBA. On Day 40, we would perform a plasmid prep for both M3 Green and M3 Red, and send them for sequencing once again.

Day 40

Inducing the expression of M3 Red & DsRed –– DNA purification from LBA –– Sending M3 Red and M3 Green for sequencing –– Confirmation gel

De-staining the gel from Day 39 confirmed our fears: the protein fractions ended up at different positions on the gel, and the ladder was smeared. Therefore, it was futile to try and identify the contaminant protein. Repeating the process of induced expression and IMAC to obtain a purer protein sample was considered a more worthy pursuit. On Day 42, when running our new protein elutions through SDS-PAGE, we would make sure to monitor the power station very closely.

On Day 39, our LBA samples were successfully inoculated with KRX (expressing M3 Red) and JM109 (expressing DsRed). Thus, having made up fresh TBA, we proceeded to induce the expression of both proteins. On Day 41, we would lyse our KRX cells to release the proteins, and purify them by IMAC.

Our cultures of KRX for M3 Green and M3 Red were also purified, and sent for sequencing. Given our previous sequencing results, however, it was prudent to run the purified plasmids on an agarose gel first. The results of this gel were as follows:

Picture22

Figure 22: Results of a 0.7% agarose gel to confirm the presence of pQE-30 carrying M3 Red and pQE-30 carrying M3 Green.

While the reader will note that the gel split in two, he will also note the presence of correct-sized bands (<4000bp) for the plasmid in all six lanes (2-7). (The bands in lanes 2 and 3 represent a small amount of plasmid that didn’t leak out. 50% Glycerol was added to allow the samples to settle in lanes 4 to 7.) Thus, we could rest assured that KRX was expressing the proteins adequately, and thus expect working sequence data.

Day 41

Lysis of KRX to release M3 Red and DsRed –– Protein purification by IMAC

Our KRX cells were lysed to release M3 Red and DsRed. A gradient imidazole wash (20, 50, 80 and 100mM) was then used to elute any and all unwanted proteins. We would check the absorbances of our fractions on Day 42.

As expected, our elutions for M3 Red were considerably brighter than those for our original DsRed:

Picture23

Figure 23: M3 Red (super-enhanced DsRed, left) compared to wild-type DsRed (right) under the transilluminator.

This result, if nothing else, demonstrates the power of mutation: how just one or two single base-pair changes can dramatically affect the phenotype of a given protein.

Day 42

Checking absorbance –– SDS-PAGE for M3 Red

Our red fluorescent protein was assumed to be either E5 (or E5up) or E57. On a spectrophotometer, the altered emission pattern that results from changing DsRed to E5up is represented by a diminished peak at 483nm (among the other 6 peaks):

Picture24

Figure 24: Fluorescence excitation and emission spectra of matured (~2 weeks at 25°C) compared to E5up. Note the diminished peak at 483 nm in the excitation spectrum after prolonged maturation. Image sourced from Terskikh et al. (2001).

By inducing the expression of DsRed and purifying it in tandem with M3 Red, we had hoped to be able to demonstrate this shift in absorbance. However, our results for DsRed were very poor. It could be that most of the protein was eluted in the washing steps:

Picture25

Figure 25: Absorbance readings for original DsRed elutions. These results would indicate that DsRed is no longer present in the solution, i.e. serious mistakes were made in the elution steps.

However, a positive result was achieved for M3 Red:

Picture26

Figure 26: Absorbance readings for our second series of M3 Red elutions. The repeat appearance of this large unknown peak – particularly after a gradient imidazole wash – prompted us to think again about this protein’s identity.

As with the results obtained on Day 38, these readings compared quite favourably with those obtained by Terskikh et al., thus reinforcing our position that the protein was either E5 (or E5up) or E57. Once again, however, there was a second peak present, similar in size to that of our protein. To confirm the presence of this unknown protein, our fractions were run through SDS-PAGE. The gel would be de-stained on Day 43.

We did not have enough time to repeat the induced expression and purification of DsRed. This doesn’t matter, however, as we know that we had DsRed to begin with; this makes obtaining the correct absorbance little more than a formality.

Day 43

Checking absorbance –– Buffer exchanging M3 Red in PBS –– Checking absorbance –– Cultures of KRX expressing M3 Green in LBA

Our de-stained gel confirmed the presence of this large contaminant protein:

Picture27

Figure 27: Results of SDS-PAGE for neat M3 Red (lane 2) and dilutions to 1 in 6 (lanes 3-7). The protein ladder did not come out well; thus, it was impossible to positively identify the large unknown proteins in each lane as dimers of M3 Red.

Thus, we buffer exchanged our M3 Red fractions in PBS and read their absorbances again. The results remained the same, however: the second peak was unaffected. Given the size of this protein, as well as the lengths to which we had purified our samples – a gradient imidazole wash to remove all proteins that were not His-tagged, along with buffer exchanging in PBS – we reasoned that it was unlikely to be anything other than a dimer or subunit of DsRed. In fact, according to Campbell et al. (2002), DsRed and its mutants have a tendency to dimerise and still fluoresce. Surely, then, the large bands on our gel could simply be dimers of M3 Red (approximately 5600bp).

However, the ladder on our gel did not come out very well: this made it difficult to be sure. Therefore, on Day 45, the gel would be rerun with two ladders.

Overnight cultures of KRX expressing M3 Green were also prepared in LBA. On Day 44, we would use these cultures to induce the expression of M3 Green.

Day 44

Inducing the expression of M3 Green –– Preparation of TBA –– Preparation of SDS Polyacrylamide gel

Using our successfully inoculated LBA samples, we proceeded to induce the expression of M3 Green. There was a need to make up 400 mL of TBA before proceeding. We also prepared an SDS Polyacrylamide gel for Day 45, in order to assess the purity of our M3 Green fractions.

Day 45

Sequencing results for M3 Green and M3 Red –– Mutagenesis PCR, round 3/3 (repeat) –– Preparation of SDS Polyacrylamide gel

The sequencing results arrived for M3 Green and M3 Red, as well as for E5. The results for M3 Red indicated that we successfully induced V71M and V105A (highlighted in green), but not S197T (highlighted in red):

Picture28

Figure 28: The amino acid sequence for M3 Red (EXPASY translation, reverse primer – reverse complement), demonstrating the induction of V71M and V105A, but not S197T.

1. As we explained on Day 23, the Alanine at amino acid 105 represents the induction of V105A.

2. The Methionine at amino acid 71 represents the induction of V71M. Our primers were designed to incorporate an Adenosine instead of a Guanine at nucleotide 211, to alter GTG to ATG; this results in Methionine instead of Valine.

3. The enduring presence of Serine at amino acid 197 reflects the absence of S197T. The induction of S197T changes Serine to Threonine: our primers were meant to incorporate an Adenosine instead of a Thymine at nucleotide 589, to alter TCC to ACC.

This suggested that our attempt at inducing S197T was a failure. However, the sequencing results for E5 indicated something else: that without knowing it, there never was any attempt to induce S197T. As shown below, V71M (highlighted in green) was present for our second mutant:

Picture29

Figure 29: The amino acid sequence for our second mutant (EXPASY translation – forward primer), showing the induction of V71M instead of S197T. The induction of V105A is highlighted in blue.

Given that V71M was meant to be our third mutation, this suggests that we induced V71M twice, without ever trying to induce S197T. We have to put this down to human error: perhaps the primers were mislabelled. This means that our second and third mutants are the same: they both carry V105A and V71M, but not S197T. Since there is no official name for this mutant, we will simply deem it “E5up with V71M”.

For DsRed, the induction of V71M, having previously induced V105A and S197T, is meant to trigger a change from red to green fluorescence. It could be that when V71M is induced in E5up without having first induced S197T, rather than the enhanced red being negated, it is merely altered slightly. If so, this would explain what was observed on Day 38: that E5up with V71M has a more pinkish fluorescence than regular E5up. (The latter is what Andrius’ protein has turned out to be, and not E57.)

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The sequencing results for M3 Green were unclear. As with M3 Red, we could see that V71M and V105A were induced (highlighted in green and blue, respectively), but not S197T (highlighted in red):

Picture30

Figure 30: Part of the amino acid sequence for M3 Green (EXPASY translation – forward primer), demonstrating the induction of V71M and V105A, but not S197T.

However, there were at least four random mutations present as well. At amino acid positions 67-69, we are supposed to see Tyrosine, Glycine, and Serine (YGS) sitting just before the Lysine (K) that precedes the sixth Methionine. These have been highlighted in yellow for M3 Red below:

Picture31

Figure 31: The amino acid sequence for M3 Red, with special emphasis on the residues preceding the Methionine at position 71.

In M3 Green, however, instead of YGS, we see Proline, Arginine, Cysteine and Threonine (PRCT). Thus, not only have Tyrosine, Glycine, and Serine been altered at random, but a fourth amino acid (Threonine) has been introduced, presumably via a frameshift mutation. Once again, this has been highlighted in yellow:

Picture32

Figure 32: Part of the amino sequence for M3 Green, showing random mutations at positions 67-70.

It is also worth noting that the full sequence is nearly divided in half over two reading frames:

Picture33

Figure 33: The complete amino acid sequence for M3 Green, divided across two reading frames.

With these abnormalities and with the absence of S197T, clearly this protein is not AG4: rather, it is likely to be a partially-ligated version of E5up with V71M, and is thus not worth purifying. Therefore, we would not proceed with cell lysis, IMAC or SDS-PAGE for M3 Green. Instead, using the time we had left, we would attempt to obtain AG4 by inducing S197T.

Mutagenesis PCR was carried out on this day, with a confirmation gel to be run on Day 46. Another SDS Polyacrylamide gel was also prepared, thereby enabling us to run two gels on Day 46 for E5up with V71M. Ideally, the first gel would show that all proteins similar in size to DsRed in the filtered lysate were indeed eluted, and are thus not represented by the large bands obtained on Day 43. (We should have run this before.)

The second gel would be used to rerun our neat protein and dilutions (to 1 in 3). This was necessary, as our ladder did not come out well on Day 43, thus preventing us from positively identifying the large protein as a dimer of DsRed.

Day 46

PCR confirmation gel –– DNA purification from PCR mixture –– Restriction digest –– SyBr Safe gel –– DNA purification from gel –– Ligation –– SDS-PAGE

The correct-sized bands were present for each annealing temperature, thus confirming the success of our PCR. Presumably, the plasmid now carries AG4. We could have achieved this earlier, had not we not wasted time inducing V71M again – that is, had we received the sequencing results for our second mutant on time.

We proceeded to purify our plasmid, carry out a restriction digest, run the digests on a SyBr Safe gel, excise and purify our best expressed plasmid, and perform a ligation. The ligated plasmid was then left with Jonathan and Donal for transformation at a later date, for by this point, we had exceeded the time allocated for the project.

SDS-PAGE was also conducted for the purposes described on Day 45. We would de-stain both gels on Day 47.

Day 47

SDS-PAGE results

On the first de-stained gel, we could see the presence of contaminant proteins in our lysate and washes, some rather large, thus bolstering the view that the unidentified bands observed on Day 43 – representing proteins that remained after the washes – were dimers of DsRed. (No image available.)

On the second de-stained gel, surprisingly, there were no large unidentified bands present with our proteins (lanes 7-9):

Picture34

Figure 34: The results of SDS-PAGE for our M3 Red elutions. Although the gel was not de-stained properly, bands could still be seen for the neat protein and the dilutions (to 1 in 3). (The angle at which the photograph was taken makes the bands appear further down the gel than they actually were.) No bands for the suspected dimers were visible on this gel.

We could see three large bands for our neat protein, our 1:2 dilution and our 1:3 dilution respectively, and virtually nothing else. Given that there had been no further washing steps since that time, along with the fact that contaminant proteins don’t just disappear, we had to conclude that the dimers separated; this may have occurred when our samples were left on the heat block.

The gel wasn’t de-stained properly, as we ran out of de-staining solution and the components to make it up (MEOH and glacial acetic acid). However, we could still see another small band for our 1:2 dilution on the gel. If this is an issue, then Jonathan and Donal still have the neat sample and the 1:3 dilution to work with.

Whatever the explanation may be, the results of this gel indicate that we did indeed obtain a pure solution of enhanced DsRed. This marked the end of the project.


Closing Statement

The pragmatic goal of this project was to obtain an enhanced form of DsRed, such that its fusion with recombinant lectins enables the easier detection of early-stage apoptosis in CHO cells subjected to stress. Not only was this goal achieved – in the form of E5up with V71M, fluorescing magenta under UV light – but the ISSC may also now have a green-emitting version of DsRed, depending on the success of our final PCR. Using the ligated plasmid obtained on Day 46, when the time is right, Donal Moynihan and Jonathan Cawley can proceed with transformation to determine if this is the case.

The broader goal of the project was to become acquainted with a series of techniques that are useful for anyone pursuing a career in practical biology. This goal was achieved and then some: for not only did we learn how to carry out site-directed mutagenesis, transformation, protein expression analysis and IMAC, but we were also given the opportunity to refine existing techniques, such as streak plating, DNA purification and gel electrophoresis.

There was a more philosophical aspect to the project as well. We learned – with great difficulty – not to panic when results go awry, as well as to expect the unexpected. We were taught that making mistakes is necessary to fully understand and appreciate any technique, be it simple or complex. These are lessons that can only be learned with experience, not from a textbook. To provide a sample of the encouragement given to us by Dr. O’Connor:

“Kareem, any laboratory that claims to have a 80 percent success rate is lying; it’s probably around 20 percent.”

Given the frequency of our mistakes, hearing this on a regular basis was most reassuring.


Acknowledgements

I would like to offer my sincere thanks to Dr. Brendan O’Connor for his almost superhuman enthusiasm for glycosylation, and for his quick replies to my queries.

I must also thank Jonathan Cawley, Donal Moynihan and Disha Choudary for their constant assistance and sage advice, in spite of the mountain of work that rests upon their shoulders.

Lastly, I would like to thank Dr. Michael O’Connell and Dr. Anne-Parle McDermott for their constant support over the past four years, in good times and bad. I am certain that without their encouragement, I would never have progressed to fourth year at all.


References

1..Campbell et al. (2007). ‘Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins.’ Biochemistry, 46: 5904 – 5910.

2..Rizzuto et al. (1995). ‘Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.’ Curr Biol. 1995; 5 (6): 635-42.

3..Ballestrem et al. (1998). ‘Actin dynamics in living mammalian cells.’ J Cell Sci. 1998; 111: 1649–1658.

4..Batisse et al., (2004). ‘Lectin-Based Three-Color Flow Cytometric Approach for Studying Cell Surface Glycosylation Changes That Occur During Apoptosis.’ Cytometry 62A: 81–88.

5..Shiratsuchi A et al. (2002). ‘Independence of plasma membrane blebbing from other biochemical and biological characteristics of apoptotic cells.’ J Biochem 132: 381–386.

6..Snapp et al. (2005). ‘Design and Use of Fluorescent Fusion Proteins in Cell Biology.’ Curr Protoc Cell Biol. Jul 2005.

7..Audouin et al. (2012). ‘Polypeptide grafted macroporous PolyHIPE by surface initiated N-Carboxyanhydride Polymerization as a platform for bioconjugation.’ Macromolecules, 45, 6127-6135.

8..Keogh et al., (2014). ‘Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa.’ Biochimica et Biophysica Acta, 2014.

9..Matz et al. (1999). ‘Fluorescent proteins from nonbioluminescent Anthozoa species.’ Nat Biotechnol 1999 Dec; 17 (12): 1227.

10..Terskikh et al. (2001). ‘Analysis of DsRed Mutants: Space Around the Fluorophore Accelerates Fluorescence Development.’ J. Biol. Chem. 2002, 277: 7633-7636.

11..Baird et al. (2000). Proc. Natl. Acad. Sci. U. S. A. 97, 11984–11989.

12..Kimata et al. (1997). Tanpakushitsu Kakusan Koso 42, 1187–1192.

13..O. Shimomura (1979). ‘Structure of the chromophore of Aequorea green fluorescent protein.’ FEBS Letters 104 (1979) 220-22.

14..Gross et al. (2000). ‘The structure of the chromophore within DsRed, a red fluorescent protein from coral.’ Proc Natl Acad Sci USA. 97, 11990-95.

15..Campbell et al. (2002). ‘A monomeric red fluorescent protein.’ Proc Natl Acad Sci USA. 2002 Jun 11; 99 (12): 7877-82.



Appendices

Appendix A: The amino acid sequence of DsRed

Picture35

DsRed is 225 amino acids in length, or 675 base-pairs. This translates roughly into a protein that is 27.5 kDa in size. The poly-His tag is fused to the N-terminal of the protein. DsRed is located within the multiple cloning site of pQE-30.

Source: Terskikh et al. (2001), ‘Analysis of DsRed Mutants: Space Around the Fluorophore Accelerates Fluorescence Development.’ J. Biol. Chem. 2002, 277: 7633-7636.


Appendix B: The nucleotide sequences of our mutagenic primers

Picture36.png

Each set of primers incorporates one SNP, to be induced in succession by mutagenesis PCR. There is a need for three sets of primers as the nucleotides to be altered in DsRed are not in close proximity to one another. A primer incorporating all three mutations would have to be at least 378 bp in length, which is obviously not feasible.


Appendix C: Table of the amino acids and their respective codons

Picture37

This table represents the redundancy of the genetic code: that is, how an amino acid may be represented by more than one codon. Leucine, for example, is coded for by six different codons. This is advantageous, for if a mutation occurs during transcription making one of those six different codons non-functional, then the other five can still code for Leucine.


Appendix D: Diagram of the mutagenesis process and primer design

Picture38

http://oxfordgenetics.com/cloning-resources/cloning-guides/site-directed-mutagenesis

Overcoming the Burqa: A Radical Approach

Had it been permissible that a person prostrates before another, I would have ordered that a wife prostrate before her husband.

Sahih Al-Tirmidhi

The Prophet said, “The women are your helpers,” which means that they are captives, and he said that a man is the “shepherd of his family and responsible for his flock.” So the singular form [of women] should be imra’a and the plural form should be nisa’, because only a man is a sayyid and women are men’s captives.

Sheikh Ibn Uthaymeen, Al-Tawhid



The burqa is designed to rob Muslim women of their individuality. As such, ninety-nine times out a hundred, the woman does not have a choice in wearing it: rather, she is forced to do so by her male relatives, whom Islam – or at least, the Wahhabi-Salafi variant – tasks with keeping women in perpetual subjugation. Their plight is comparable to that of the Jews under Nazi Germany, who were forced to wear the Star of David as a mark of inferiority. It’s not a case of freedom of religion, despite the naïve assumptions of Western commentators and shady assertions of ‘liberal Muslim’ apologists.

Indeed, when we examine the claims of so-called moderates within the mainstream faith, they often prove to be just as sinister as the extremists they so condemn. One example would be Tariq Ramadan, who has called for a “moratorium” on stoning people to death, rather than its abolition. It as Sigmund Freud once said: where questions of religion are concerned, people are guilty of every possible sort of dishonesty and intellectual misdemeanour. To quote Latifa, author of My Forbidden Face, on the degradation and physical suffering of being made to wear the burqa under the Taliban:

I look at this garment, its woven cloth flowing all the way down to the ground from a closely-fitted bonnet which completely covers the head… It’s suffocating. The cloth sticks to my nose. I have a lot of trouble adjusting the embroidered lattice slits in front of my eyes… In order to turn my head, I have to keep some of the cloth clutched beneath my chin so that the eyeholes stay in place. In order to look behind me, I have to turn round completely. I can feel the rustle of my own breath inside the garment. I’m hot. My feet get tangled up in the material. I’ll never be able to wear this. I now understand the stiff robot-like walk of the ‘bottle women’. I now know why they hesitate for so long before crossing the street, why it takes them an eternity to walk upstairs. These phantoms that now roam the streets of Kabul have a terrible time avoiding bicycles, buses and carts. It’s even worse trying to run away from the Taliban. This is not a garment. It’s a moving prison… I climb out of the burqa feeling humiliated and furious. My face belongs to me. The Qur’an says that a woman can be veiled, but that she must remain recognisable. The Taliban want to steal my face, forbid us all our faces.

The issue of the burqa, therefore, is inherently one of women’s rights. Thus, many people in the West feel moved to ban it outright. I believe the sentiment behind this to be correct: for certainly, we shouldn’t allow this sort of backwardness to take root here. In practice, however, banning the burqa does nothing to combat the oppression of Muslim women: on the contrary, it only acts as an incentive for their husbands and fathers to keep them at home, where beatings and rape are commonplace. It is to tackle the symptom, not the problem itself. I thus propose a more comprehensive solution, if a radical one:

1..Enact legislation outlawing Wahhabi-Salafism as a violent political ideology (analogous to Nazism), being the common denominator of ISIS, Al-Qaeda, and the Taliban;

2..Have all grown men who appear to be of this tendency – cleric, teacher, and layman alike – identified as such, and subsequently detained to assess the sincerity of their beliefs;

3..Use neuroscientific techniques such as fMRI1 (once the technology has caught up2) to identify those who give their wives a choice in wearing the burqa, with all those who don’t being deported to Saudi Arabia;

4..Have the State provide for all wives and daughters that are left behind, in the hope that they will abandon the burqa of their own accord.

Now of course, it is inevitable that many of these women will double down on their faith, interpreting this moral intervention as a Western conspiracy against Islam. We have to allow for this, for some people just aren’t as bright as others. With enough time, however, the joys of suffrage ought to win through – or at least, one would hope so. If, after years of exposure to the equality and free expression provided by liberal democracy, the majority of these women still insist on wearing the burqa, then we absolutely would have to ban it, with those who defy this ruling proving so indoctrinated as to be quite beyond redemption.

In such a scenario, we would have little recourse but to extend them the same courtesy as their male relatives, i.e. deportation. Until then, however, we are justified in viewing them as the victims of religious barbarism. Let us work towards a humanist government that will punish those who treat women as slaves, with a special intolerance for those who substitute secular law for desert scripture. In the case of the burqa, this starts with challenging the climate of intellectual fascism that has developed around Islam and Muslim culture, enabled by many on the Left. To finish with a quote from Ayaan Hirsi Ali, ex-Muslim activist and author of The Caged Virgin:

The reason I am determined to make my voice heard is that Muslim women are scarcely listened to, and they need a women to speak out on their behalf. Their official spokespersons are nearly all men. Given the widespread suffering of Muslim women, there are too few social organisations and political parties actively devoted to improving their lot. Spokesmen of Muslim organisations and immigrant politicians with Muslim backgrounds, along with other advocates of “group rights”, excel in denying, trivialising, or avoiding the enormous problems of Muslim girls and women in the West.

In a June 2002 interview, the member of parliament for the Socialist Party, Khadija Arib, said the following about the position of Muslim women: “People seem to think that immigrant women want to sit home alone all day, but this happens mostly because there is nowhere for them to go.” At the opening in spring of a mother-and-child daycare center in an Amsterdam suburb, she proposed establishing a special facility where women could attend activities all day long. In doing so, she denies the essence of the problem. In a large segment of the Muslim community, the notion still exists that women should not have any freedom of movement or work outside the home. Muslim women will benefit more from harsh criticism of this idea than from the creation of special women’s activity centers

Multiculturalists take no heed of the private lives of the cultures they are defending. And it is precisely in private life that differences in power and the repression of women manifest themselves most clearly… I invite the advocates of the multicultural society to acquaint themselves with the suffering of the women who, in the name of religion, are enslaved in the home. Do you have to be mistreated, raped, locked up, and repressed yourself in order to put yourself in someone else’s position? Is it not hypocritical to trivialise or tolerate those practices, when you yourself are free and benefit from mankind’s progress?

A multicultural society is not a goal in itself. We in the West need to make a concerted effort to counter Islamic education and all those other Islamic institutions that lead to self-segregation and thus contribute to the continuation of a hopeless tyranny over women and children.


Footnotes

1. This is an acronym for functional Magnetic Resonance Imaging. Over the last decade, scientists have used fMRI to locate regions of the brain that change when a person lies. This technique measures changes in blood flow in the brain – a reflection of neural activity – as people answer questions while inside of a scanner. The resulting images pinpoint brain activity in specific regions during the lie and truth phases of the deception paradigms.

Although several brain areas appear to play a role in deception, the most consistent finding across multiple fMRI studies is that activity in the prefrontal cortex increases when people lie. The prefrontal cortex, situated just behind the forehead, is a collection of regions responsible for executive control, i.e. the ability to regulate thoughts or actions to achieve goals. Executive control includes cognitive processes such as planning, problem solving, and attention. These are all important components of deception, so it is no surprise that the prefrontal cortex is active when we lie. Dishonesty requires the brain to work harder than honesty, and this effort is reflected by increased brain activity. Studies even show people take longer to respond when lying.

2. We are still a long way from using fMRI to identify deceit outside of the laboratory. One major obstacle concerns countermeasures, e.g. small movements, changes in breathing, and altered cognitive processing. By using countermeasures, a person may be able to deliberately offset any brain changes associated with deception to defeat lie detection technology. A recent study found the accuracy of fMRI for lie detection dropped to a mere 33 percent when participants used countermeasures during questioning.



A Syrian woman casts off the niqab, having been liberated from the tyranny of Daesh.

Applying Science to Right-Wing Views of Addiction & Poverty

To blame the poor for subsisting on welfare has no justice, unless we are also willing to judge every rich member of society by how productive he or she is. Taken individual by individual, it is likely that there’s more idleness and abuse of government favours among the economically privileged than among the ranks of the disadvantaged.

Norman Mailer



Prologue

An Excerpt from Sociology: The Essentials, 9th Edition

Pages 194 – 195:

Public debate about poverty hinges on disagreements about its underlying causes. Two points of view prevail. The first view, popular with the public and many policymakers, is that poverty is caused by the cultural habits of the poor, i.e. that they are to blame for their own condition. The second view is more sociological, one that understands poverty as rooted in the structure of society, not in the morals and behaviours of individuals.

Blaming the poor for being poor stems from the myth that success requires only individual motivation and ability. Many in the United States adhere to this view and hence have a harsh opinion of the poor. This attitude is also reflected in U.S. public policy concerning poverty, which is rather ungenerous compared with other industrialised nations. Those who blame the poor for their own plight typically argue that poverty is the result of early childbearing, drug and alcohol abuse, refusal to enter the labour market, and crime. Such thinking puts the blame for poverty on individual choices, not on societal problems.

The ‘culture of poverty’ argument attributes the major causes of poverty to the absence of work values and the irresponsibility of the poor. In this light, poverty is seen as a dependent way of life that is transferred, like other cultural values, from generation to generation. Policymakers have adapted the culture of poverty argument to argue that the actual causes of poverty are found in the breakdown of major institutions, including the family, schools, and churches.

Is the culture of poverty argument true? To answer this question, we might ask: Is poverty transmitted across generations? Researchers have found only mixed support for this assumption. Many of those who are poor remain poor for only one or two years; only a small percentage of the poor are chronically poor. More often, poverty results from a household crisis, such as divorce, illness, redundancy, or parental death. People tend to cycle in and out of poverty. The public stereotype that poverty is passed through generations is thus not well supported by the facts.

A second question is: Do the poor want to work? The persistent public stereotype that they do not is central to the culture of poverty thesis. This attitude presumes that poverty is the fault of the poor, that poverty would go away if they would only change their values and adopt the American work ethic. What is the evidence for these claims?

Detailed studies of the poor find no basis for the assumption that the poor hold different values about work compared to everyone else (Lakso, 2013; Lee and Anat, 2008). They simply find that work is difficult to obtain. Several other facts also refute this popular claim. Most of the able-bodied poor do work, even if only part-time. The number of workers who constitute the working poor has actually increased. You can see why this is true when you calculate the income of someone working full-time for minimum wage: someone working forty hours per week, fifty-two weeks per year, at minimum wage will have an income far below the poverty line. This is the major reason that many have organised a living wage campaign, intended to raise the federal minimum wage to provide workers with a decent standard of living.

Current policies that force those on welfare to work also tend to overlook how difficult it is for poor people to retain the jobs they get. Prior to welfare reform in the mid-1990s, poor women who went off welfare to take jobs often found they soon had to return to it because the wages they earned weren’t enough to support their families. Leaving welfare often means losing health benefits, yet incurring increased living expenses. In sum, attributing poverty to the values of the poor is both unproven and a poor basis for public policy.



Article

Politics, dear reader, is all about smoke and mirrors. When Theresa May speaks of creating a United Kingdom that “works for everyone, not just the privileged few”, she is consciously trying to distract people from the heart and soul of the Conservative Party, which exists precisely for, and is run by, the privileged few. From the savage treatment of striking miners to the wholesale privatisation of the NHS, Toryism has consistently defined itself as the enemy of working class Britons. We infer the Tories’ attitude towards the poor from their policies – see the ‘Fit for Work’ scheme, which punishes the disabled by having them work jobs that are well beyond them1 – for the purpose of policy is to further party ideology.

When Fine Gael cut lone parent’s allowance all the while enabling billionaires to pay no tax, this betrays their stance towards the most vulnerable in Irish society – namely, that they deserve to suffer. Indeed, if there is one thing history has shown, it is that right-wing politics is rooted in the survival of the fittest. It is a very callous way of thinking, absolving individuals of the responsibility to help those in need. Naturally, those who embrace this mindset have no trouble subverting science to advance their agenda. Addiction has nothing to do with the brain, argues Peter Hitchens, but is purely a matter of willpower. Social welfare is to blame for the low socioeconomic status of blacks in America, alleges Thomas Sowell.

It takes a curiously judgemental disposition, one that naturally leads people into right-wing politics, to make such austere claims about our species’ condition. It certainly doesn’t follow from an awareness of modern science, which impels us to dispense with such claims. With regards to addiction, recent developments in neurogenetics have demonstrated how the propensity to alcoholism, far from being a choice or moral problem, is determined largely by mutations in a heterogeneous collection of genes located within the prefrontal cortex; indeed, the genetic liability in humans is estimated to be between 50 and 60 percent (Dick and Bierut, 2006). To quote from a comprehensive study by Farris et al. (2015):

Numerous genes are known to affect alcohol consumption and other alcohol-induced behavioural phenotypes in animal models (Crabbe, Phillips, Harris, Arends, & Koob, 2006). Mirroring findings from preclinical models of alcohol-related traits, human genetic studies suggest that a heterogeneous collection of genes influence the risk of developing Alcohol Use Disorder (AUD)… Genetic risk factors may not directly translate into psychiatric diagnosis, but rather contribute to intermediate molecular and behavioural phenotypes tied to disease states. Transcriptome information is processed directly from the DNA template, serving as one of the most proximal molecular traits influenced by genetic variation, with external forces also acting to regulate cellular responses.

The analysis presented by Dr. Sean Farris tested the hypothesis that the expression of systematic gene networks was disrupted within the brains of alcohol-dependent individuals. RNA-Seq was used to profile total RNA from the postmortem prefrontal cortex (PFC), a key region in alcohol abuse and addiction (Goldstein & Volkow, 2011). Human brain samples, obtained from the New South Wales Research Center (Sheedy et al., 2008), were thoroughly matched for multiple variables, including age, brain weight, post-mortem interval, and RNA integrity… The average number of significant connections per transcribed genomic feature was markedly reduced in alcohol-dependent brain tissue, indicating a loss of homeostatic transcriptome-wide regulation. Compensating for a loss in overall network structure, subsets of genes are packaged into more densely connected clusters that presumably underlie addictive processes.

Weighted gene coexpression network analysis of the alcoholic and matched control subjects discerned a total of 38 and 32 gene modules, respectively (Farris, Arasappan, Hunicke-Smith, Harris, & Mayfield, 2014). Modules were similar to previous network studies of brain tissue, reflecting the organisation of gene expression in defined regions of the CNS (Oldham et al., 2008)… Those modules within the upper-quartile related to lifetime alcohol consumption could be further distilled into two main groups (Group1 and Group2) based on inter-module correlations, while the lower-quartile gene expression modules showed no overt similarities (Farris et al., 2014).

The voltage-gated sodium channel type IV beta subunit (Scn4b) is a suspected quantitative trait gene for alcohol drinking behaviour in mice (Mulligan et al., 2006; Tabakoff et al., 2008). Furthermore, SCN4B, a highly connected gene within Group1 of alcoholic gene modules, was significantly correlated to the lifetime amount of alcohol consumption… Genes residing within Group1 of coexpression modules (Farris et al., 2014) were strongly enriched for human synaptic protein complexes (Bayés et al., 2011). Acting as a representative map for probable interactions, the human gene expression networks provide a valuable resource, using only a small number of samples, for exploring specific isoforms and protein complexes related to chronic alcohol effects.2

Science teaches us that the brain actually changes with addiction; and thus, that it takes far more than willpower or good intentions to fully overcome it. Neural imaging studies of individuals addicted to drugs or alcohol show decreased activity in the prefrontal cortex, which is the region responsible for making decisions. When the prefrontal cortex isn’t working properly, it is virtually impossible to stop taking the drug – even if the victim is aware that he might lose custody of the children for doing so, or possibly end up serving a prison sentence. To quote from a study by Ducci and Goldman (2012), examining neuroadaptive changes resulting from prolonged drug abuse:

In individuals who are vulnerable to addiction, repetitive exposure to the agent induces long-lasting neuroadaptive changes that further promote drug-seeking behaviours and ultimately lead to persistent and uncontrolled patterns of use that constitute addiction. These neuroadaptive changes are the bases for tolerance, craving, and withdrawal and lead to a motivational shift. Motivation to drug-seeking behaviour is initially driven by impulsivity and positive reward. In contrast, compulsivity and negative affect dominate the terminal stages of the pathology. Addictions are in a sense “end-stage” diagnoses, because at the time diagnosis is made, potentially irreversible neuroadaptive changes have occurred – changes that were preventable at an early point of the trajectory of the illness.3

Taking all of this into account, it is clear that the likes of Peter Hitchens are way out of their depth. The contrast between raw scientific data and the speculative ramblings of a Little Tory is a stark one. There is any number of papers and textbook chapters explaining the biology of addiction, all of which are available to the public. And yet, Hitchens continues to argue not only that addiction is a myth, but that law enforcement is the solution to drug abuse. In an interview with The Guardian, Hitchens comments as follows:

No, it’s just laughable. I believe in free will. People take drugs because they enjoy it…. All these people would be helped by a properly enforced law which punished them for doing it, because then fewer would do it, and they’d be rescued from it.

While it is true that many people take drugs such as cannabis because they like it, doesn’t Hitchens wonder why those same people would never dream of touching heroin? Happy, successful, stable people seldom inject heroin, whereas most junkies have suffered catastrophic childhoods, often in care homes. And if the horror of heroin addiction is insufficient to deter someone from shooting up, how is the prospect of being arrested any more likely to put them off? Hitchens places unfathomable faith in the power of the law to control human behaviour.


Sowell Searching

We will now address the low socioeconomic status of blacks in America, or at least attempt to do so. To avoid making a straw-man argument, let us step back to examine precisely what Thomas Sowell is saying. Having read the titular essay of Black Rednecks and White Liberals, I can safely state that Sowell’s argument is threefold:

1. That “ghetto culture” is an offshoot of white redneck culture which existed in the antebellum South, i.e. prior to the Civil War;

2. That this tendency has since become a major cultural influence on black people in America; and

3. That for blacks in America to prosper, they must forsake their violent culture, receive no government help, and copy the successful white people around them. In Sowell’s words:

White liberals, instead of comparing what has happened to the black family since the liberal welfare state policies of the 1960s were put into practice, compare black families to white families and conclude that the higher rates of broken homes and unwed motherhood among blacks are due to “a legacy of slavery”. But why the large-scale disintegration of the black family should have begun a hundred years after slavery is left unexplained… External explanations of black-white differences – discrimination or poverty, for example – seem to many to be more amenable to public policy than internal explanations such as culture… Such external explanations require no painful internal changes in the black population but leave all changes to whites, who are seen as needing to be harangued, threatened, or otherwise forced to change.

Now as regards the first point, I think Sowell makes a rather convincing case. It is fascinating to learn how the development of Southern culture was influenced, however slightly, by ruffians from the Celtic hinterlands. The similarities between ghetto culture – as defined by an aversion to work, drunkenness, sexual promiscuity and proneness to violence – and white redneck culture are striking, to say the least. It is clear that the two are linked in some fashion, even if this is not objectively demonstrable.

It is when we reach the second point of Sowell’s thesis that it begins to break down. Sowell argues that ghetto culture is a major cultural influence on blacks in America, i.e. that having supplanted the remnants of African cultures in the antebellum South, ghetto culture began spreading to more urban areas:

Much of the cultural pattern of Southern rednecks became the cultural heritage of southern Blacks, more so than survivals of African cultures, with which they had not been in contact for centuries. Such cultural traits followed Blacks out of the Southern country sides and into the urban ghettos where many settled.

Now for redneck culture to have had such an impact, dear reader, there would need to have been a strong presence of Celtic ruffians in the Southern states. However, judging from the data available to us, this was simply not the case. Sowell cites Cracker Culture by the fringe scholar Grady McWhiney to support his position, but census data for Greene County during this period refutes McWhiney’s claim that Mississippi’s Piney Woods region was predominantly Celtic: rather, surname analysis indicates that most settlers were English, and that all settlers were at least one generation removed from their home country4.

Furthermore, while Sowell does acknowledge that ghetto culture does not characterise most black Americans5, he nonetheless argues that it is a major influence on the black population. As evidence of this, he points to the fact that over a quarter of black Americans are currently living in poverty (27 percent6), i.e. that this reflects habits of improvidence on their part. But as we saw earlier, being poor has little to do with having a poor work ethic. Of that 27 percent, it is reasonable to assume that majority simply find it difficult to get work.

We now come to the final point of Sowell’s thesis, concerning violence, welfare provision and the perceived white work ethic. First of all, while violence in black communities is certainly a major problem, Sowell’s thesis runs contrary to long-established principles of cause and effect. Violence doesn’t create and perpetuate poverty, but poverty can create and perpetuate violence: the antebellum South was very prosperous despite having one of the most violent systems ever conceived (slavery), whereas the correlation between long-poor populations and high rates of violence is omnipresent in the world. Populations don’t start off rich, begin tolerating violence and thus become poor – that’s not how it works, as any basic textbook on sociology will affirm.

Secondly, it is wrong to suggest that by cutting off black children from food stamps, housing assistance and public schools, this would produce a more prosperous generation who are “hungrier for success”. Anyone who has spent time in Africa, South America or the Arab world knows that the absence of social welfare and affordable education inevitably leads people into crime, including drug peddling and prostitution. All too often, it leads to mass exploitation by criminal enterprise: see Qatar’s use of slave labour to build hotels and stadiums by way of Lee Trading Ltd. To quote from a major sociological analysis of twenty European countries, showing how the introduction of social transfers (benefits) has been the most effective way to reduce in-work poverty (Lohmann, 2009):

Table 3 shows the incidence of in-work poverty and the extent of poverty reduction via social transfers in all of the 20 countries which are examined in this paper. For better orientation, the countries are grouped regionally, which produces a strong overlap with broadly used welfare state typologies… In all countries including the Southern European countries, the post-transfer rate is lower than the pre-transfer rate. In addition, Table 3 reports the relative degree of poverty reduction which is often more adequate because in absolute terms, poverty reduction can be high simply because we start off from a higher level of pre-transfer poverty.7

Pic 1

Indeed, it takes a wilful ignorance of industrial neglect to believe that there is this ocean of jobs which black Americans refuse to take. The city of Detroit, where black Americans comprise over 84 percent of the total population, is a perfect example of this. In 1950, Detroit was a manufacturing machine, with a workforce of nearly 300,000 people. Today, due to large-scale abandonment by the private sector, that number is closer to 25,000. To avoid having to abide by progressive labour laws, companies such as Honda and Toyota closed up shop and moved to the South; this caused an economic collapse, with over 1.1 million people having since left the city to find work. Without jobs, there can be no employment; and without employment, there can be no tax base to help bring Detroit back to life.

Pic 2

(The remains of the old Packard factory, which shut its doors in the late 1950s.)

Lastly, we must state that emulating the behaviour of whites is not a sure-fire way to the top. Presumably, the quality that Sowell would most like to see among black Americans is a strong work ethic. But there are numerous studies showing how, in order to able to compete, black people are forced to work harder than their white counterparts. To quote from a recent paper by the National Bureau of Economic Research (Cavounidis and Lang, 2015):

We develop a model of self-sustaining discrimination in wages, coupled with higher unemployment and shorter employment duration among blacks. While white workers are hired and retained indefinitely without monitoring, black workers are monitored and fired if a negative signal is received. The fired workers, who return to the pool of job-seekers, lower the average productivity of black job-seekers, perpetuating the cycle of lower wages and discriminatory monitoring.8

Pic 3

While it is true that blacks in America have traditionally had a lower skill-set than whites, this in no way suggests that their work ethic isn’t on par. It does mean, however, that while equality of opportunity remains the central issue, it perhaps doesn’t tell the whole story.  To quote Cavounidis and Lang (2015) once more:

Our model also strongly suggests that history matters and that equality of opportunity is not enough to eliminate racial disparities in the labour market even if this concept is used very expansively. The fact that blacks historically had low skills leads to an equilibrium in which the pool of black job-seekers has lower skills than the pool of white job-seekers, even when the distribution of skills among all workers is identical for blacks and whites.9

I will conclude with this, dear reader. Reason dictates that for any writer to be taken seriously on the socioeconomic divide in America, he must first recognise that it is a multi-faceted issue. To reduce it to a matter of “welfare making people lazy” is not becoming of any earnest seeker of truth. While such an approach may be convenient for right-wing pundits to take, it has absolutely no place in the field of science. The difficulties in being able to afford a decent education10, the incarceration of young black men in prisons run for profit11, the existence of genetic differences in brain size12 – each of these plays a major role in keeping black Americans at a disadvantage, commanding sympathy from those of us with a shred of humanity.



Footnotes / References

1. According to the Royal College of Psychiatrists, ‘Fit for Work’ tests are causing relapses in patients with serious mental health conditions. As of November 2015, public health researchers at the University of Liverpool have linked the assessment to 590 suicides, as well as 279,000 cases of self-reported mental health problems and 725,000 anti-depressant prescriptions. Consider the case of Mark Wood, who died in February 2014. Despite struggling with multiple mental health problems, with his doctor describing him as “extremely unwell and absolutely unfit for any work whatsoever”, the 44-year-old was found “fit for work” in 2013. Four months later, he was found dead in his home weighing just 5st 8lb.

2. Farris et al. (2015): ‘Applying the New Genomics to Alcohol Dependence’, Alcohol.

3. Ducci and Goldman (2012): ‘The Genetic Basis of Addictive Disorders’, Psychiatr Clin North Am.

4. Cherilyn A. (1998): ‘Grady McWhiney’s “Antebellum Piney Woods Culture”: the Non-Celtic Origins of Greene County, Mississippi’. Journal of Mississippi History.

5https://newpittsburghcourieronline.com/2017/01/22/black-rednecks-and-affluent-black-assumptions/2/

6. http://www.npc.umich.edu/poverty/

7. Lohmann, Henning (2009): ‘Welfare States, Labour Market Institutions and the Working Poor: A Comparative Analysis of 20 European Countries’. European Sociological Review.

8. Cavounidis and Lang (2015): ‘Discrimination and Worker Evaluation’. Boston University, NBER and IZA.

9. Ibid.

10. In 1971, Harvard’s $2,600 tuition amounted to about thirteen weeks’ worth of the median household’s annual income. Today, the median household needs to work for almost a year to pay the full sticker price. To make up the difference, millions of students and families are forced deeper into debt every year: indeed, it is estimated that $100 billion a year is borrowed through a cottage industry of private and publicly-funded loan programs.

11. See the recent documentary 13th by Ava DuVernay, examining the evolution of slavery in the United States since abolition, from convict leasing to mass incarceration:

https://en.wikipedia.org/wiki/13th_(film)

12. See the following study by J. Philippe Rushton and Arthur R. Jensen, examining racial differences in cognitive ability over a thirty year period (1975 – 2005):

https://www1.udel.edu/educ/gottfredson/30years/Rushton-Jensen30years.pdf

Statement on Travellers, SJWs & Ex-Muslims of Ireland

What liberals mean when they talk about “respect” is that they can handle complex fiction, ambiguity, and criticism, but that other people can’t, especially people in minority communities, because they are too sensitive.

Monica Ali


This is a very brief comment on the troubled relationship that exists between this blog and Ex-Muslims of Ireland (EMI), incited primarily by militant Social Justice Warriors on Twitter. Essentially, these people attempted to discredit the EMI using an offensive piece I had written on this blog four years ago. The article portrayed the Travelling community in a very negative light, and was thus sufficient for their purposes.

To be clear: this was a fearful, bewildered response to having my life threatened by someone who happened to be from that community. In struggling to manage the stresses of college and domestic life, in that instance, it was easier for me to submit to emotion and prejudice than to think rationally.

Having since revised my views about Travellers, whose integration into wider Irish society has been seriously hampered by austerity cuts, I now deeply regret writing that piece. I especially regret the dehumanising language that was used: indeed, given the racist abuse that my father has often endured, I really should have known better. I have thus apologised for it and removed it from my blog; we have also issued a statement on the website to this effect.

None of this, however, has stopped SJWs from trying to discredit us. Indeed, I now have political allies asking me about the piece, demanding to know my views on the Travelling community. Having already apologised, I feel that there is nothing to explain. If anything, it is those who help keep Muslims enslaved to dogma who have the explaining to do. Those who suggest that Muslims are so intellectually deficient that they must be sheltered from all criticism – these are the people who need to explain themselves, not me.

Kareem