Poster Presentation at LIYSF

A member of Oxford iGEM presented their idea in the Bazaar at the London International Youth Science Forum (LIYSF), which took place from 27^th July to 10^th August. LIYSF celebrates its 58 years of history, and has been granted UNESCO patronage this year. The forum involves lectures from pioneering researchers, visits to research centres in the UK, and a rich engagement and exchange of ideas amongst participants. 475 representatives from 75 countries participated, and over a hundred science projects were presented.

In our presentation, we discussed the importance of finding alternative treatments for Wilson’s disease and raising public awareness towards rare disease. Most of the participants were unaware of Wilson’s disease, and they showed interest towards the issue. One student from Poland, however, was diagnosed with Wilson’s disease, which was later proven to have been false-positive. He showed genuine interest towards this issue, being aware of current treatments, and was keen to see improvements on both treatment and diagnosis.

Meeting with Professor Jane Kaye

Professor Jane Kaye is a Professor of Health, Law and Policy and the Director of the Centre for Health Law and Emerging Technologies at the University of Oxford. Her work focuses on reconciling new medical technologies with current laws and ethics. In our meeting with her we aimed to use her expertise to gain further insight into the legal and ethical issues our project raises.

As we have found through our surveys, the main issues she thought there would be with the project were environmental and patient safety. In order for our project to progress further we would need to ensure that the bacteria could not take over the gut or survive in the environment. We propose to have a “kill switch” which would enable us to kill the bacteria if necessary so that they could not survive outside the body. She said the public lack confidence in GMOs but ultimately it is up to the patients themselves to decide whether to take the risks or not. From our discussions with patients at the Wilson’s conference it seems that the patients themselves would be willing to try a new treatment.

Our survey showed there was some religious objection to synthetic biology, the idea that we are “playing God” whilst designing our bacteria. She thought there wasn’t really a way around this as what we are doing wouldn’t have happened naturally (unlike with plant GMOs where evolution is being sped up) so instead we should address this by focussing on the huge benefits this sort of treatment could have. She added that we should not expect to be able to win everyone over but that differences in opinion can be a good thing to ensure that scientists continue to question their work.

Professor Kaye emphasised the importance of continual engagement with the public as opinions will change over time and it is important to use that to direct our work. At key points she recommended going back to the patients themselves and asking them which we are able to do through their Facebook page, but also making sure not to overwhelm them by asking them too often. It was her view that while the public should be consulted and involved in decision making ultimately it is the scientists themselves that will make the decisions. It was very useful to see how we could better integrate some of our human practices into our project and some of the ethical issues can be addressed from a very knowledgeable source.

Meeting with Professor Kevin Foster

Kevin Foster is a Professor of Evolutionary Biology in the Zoology department of Oxford University who we met with to discuss gut bacteria in relation to our project.

We asked if our idea of creating a stable E coli population in the small  intestine was feasible. He said it might be but E coli are not actually that common in the small intestine and are confined mainly to the colon. Bacteriodes or lactobacillus were suggested as a more abundant species but these would be difficult for us to work with as they are anaerobic.  However, he agreed that if we were able to prove that our idea worked in E Coli we may be able to transfer this into a species that could survive in the small intestine.

He mentioned faecal transplants as a way in which bacteria have been introduced to the gut but as these bacteria are already adapted to the intestines these wouldn’t have the disadvantages that ours would have. In order for our bacteria to be able to persist in the gut they would have to be able to outcompete those that are already present whilst using a large amount of energy to make our copper chelator. His solution to this was to “arm” our bacteria to make them competitive against the other species present.

Our bacteria could be made competitive by adding colicins to them. These contain genes used to attack each other – “battle strains”. They have toxin and anti-toxin components. A portion of the cells with the plasmids lyse to release the toxin. This lysis kills the cells so some of the population will die as they kill other cells but the ones that don’t lyse survive as they can create the antitoxin. Colicins could potentially be used to remove the existing E Coli in the gut to enable our engineered E Coli to fill their niche.

It was very helpful for us to meet with Professor Foster as it enabled us to identify some of the potential issues we might face in our project so we could adjust our approach before we started working in the wet lab.

Meeting with Dr Garry Brown

Oxford iGEM members had a meeting with Dr Garry Brown, a University lecturer in the Department of Biochemistry. We were in a lucky position to gain expert advice in the early stages of our project from a doctor who had previously worked with patients suffering from Wilson’s in his career. This short blog post details what we have learnt from interviewing him about our project.

Penicillamine was used 30 years ago, and is still used. Patients complain about the current treatment, so people are working on alternatives – and all are based on chelation methods.

Wilson’s disease manifests at different ages. As a baby, an acute presentation would be liver disease in the newborn. As an adult, Wilson’s generally manifests through neurological and psychiatric symptoms, chronic evolving diseases and schizophrenia in the teenage years. Therefore, there is a need for a quick-working therapy in the short term acute case, as well as life-long chronic treatment. Dr Brown suggests that whilst our treatment would not be suitable for a severe, acute case, it has potential as a long-term therapy, which is what we intended for it to be.

Dr Brown told us that there is growing interest in the science of repopulating the gut flora.  An interesting application of potential benefit is for Caesarian-born babies, who have a different gut content.

Dr Brown agreed that in an ‘in vitro’ (test-tube) experimental approach in our project, we should focus on using E. coli, and then suggest the bacterial species we would transfer and adapt our BioBricks into which would be more prevalent in the area of the gut we are targeting.

Conclusion:

“The therapies used today are the same as those in the 1980s – zinc and copper chelators. The chelators DO work, but with severe side effects, and there has been little interest in research looking new treatment methods since.”

 

Oxford iGEM @ 2016 Wilson’s Disease Support Group – UK Meeting & 6th AGM

On Sunday 24th July, Oxford iGEM attended the 6th Annual General Meeting (AGM) of the Wilson’s Disease Support Group (WDSG), based in Cambridge. We were privileged to be invited to give a presentation about our project, and to speak to patients with Wilson’s and learn about the disease first-hand, in order to learn more about their conditions and how our therapy might be of benefit to them.

Sam, Shu and Andreas attended the annual Wilson’s Disease Support Group  in Cambridge

Our Presentation

We delivered a presentation at the conference about our team and how we are using synthetic biology to demonstrate the proof-of-concept for our treatment. A lot of patients were really interested in our talk and agreed that a new treatment was in need. Rupert Purchase, the President of the Wilson’s Disease Support Group, was particularly interested in the chemical composition of our protein chelator.

Andreas presenting our team’s idea as a possible treatment for Wilson’s in the future

Talking to Patients

The Wilson’s disease patients we met at the society were friendly and happy to talk about how their conditions affected their day-to-day life. It was noticeable that some people looked almost perfectly recovered, having taken Penicillamine for decades, whereas other patients had speech impediments. Where Wilson’s disease symptoms occur first depends on the individual – typically either first in the brain (symptoms include speech impediment, uncontrollable shivering similar to Parkinson’s disease, dribbling) or in the liver. How they occur also depends – some people’s condition gradually worsen when they stop taking pills, whereas others may suddenly present with severe illness when copper levels build up to a critical amount.

 

There were main 3 issues with current treatment:

• Side effects
• Need for constant administration at certain times; need to be refrigerated
• Too expensive

Andreas talking to Martha, the representative of the equivalent Wilson’s Disease Group in Poland

Toxicity and quality of life

Penicillamine is strong and can have severe side effects, where Trientine is milder and adequate for early stage treatment. Because of the NHS policy, however, doctors usually administer Penicillamine first. One patient experienced severe illness including vomiting as side effects – after which the doctor switched her therapy to Trientine.

One patient was initially taking Penicillamine but was taken off it due to developing a skin rash. They are now taking 4 pills of Trientine daily. Once they were diagnosed, the doctors immediately checked their sibling, who was diagnosed with Wilson’s as well. Since the sibling didn’t have any symptoms, the doctors started zinc therapy, which is supposed to block absorption of copper in the body. Since then, the sibling has been taking zinc and has no visible side effects from either her condition or the zinc therapy.

Patients commented on the difficulty of getting employment due to Wilson’s disease. Companies do not even consider employing patients, and public awareness is low, with there only being one patient with Wilson’s in many counties. In other cases, patients whose symptoms worsened as a result of either penicillamine or the disease have often had to leave their jobs and struggle financially as a result.

Cost of current drugs

Until 2014, Trientine used to cost £480 for 100 capsules, each containing 300mg. Patients are required to take four capsules a day, morning and night. Now, the cost has increased to £3,353, seven times of its original price. Similarly, the price of Penicillamine has also increased. These pills are licenced by a company called Univar, who used to sponsor WDSC until last year. Univar claims that the price raise is necessary, but the chemical process of creating Trientine is supposed to be cheap. At the moment, the parliament and NHS doesn’t have the authority to sue or question companies for their drug pricing, and since all Wilson’s disease treatment is monopolised by Univar (apart from the US, where the price is even worse, $22,000), Wilson’s disease patients have increasingly limited access to their treatment. As Penicillamine is cheaper than Trientine, and the licence for Trientine states that it should be treated only to patients with intolerance towards Penicillamine, NHS’s policy is to first treat patients with Penicillamine.

One patient was initially given penicillamine (6 tablets/day) when he was diagnosed, but eventually developed lupus. His treatment was changed to trientine (4 tablets/day) but eventually stopped taking it because it was too expensive and he and his wife could not afford it; it also gave him cramps in hands and legs. He is now back on penicillamine but on a lower dose (4 tablets/day). He has a slight tremor on his left hand due to copper affecting his brain. He also speaks slowly but other than that he seems completely normal.

Awareness of Wilson’s Disease

Patients generally thought that there was low awareness of Wilson’s Disease within the medical community in the UK, causing later diagnosis and greater liver/brain damage before treatment can begin. One patient expressed their disappointment that they would end up explaining what Wilson’s disease is to doctors, and wished that doctors would be more aware of rare diseases in general (even if they might not be knowledgeable about them). There are a few experts in Wilson’s disease in the NHS, but not enough communication is made within the NHS.

The siblings of patients who showed Wilson’s disease symptoms are immediately DNA tested, and these patients are diagnosed to have Wilson’s disease before symptoms appear. Sometimes it is the opticians who discover symptoms of Wilson’s disease before doctors do. Many patients, however, are less fortunate to be diagnosed to have Wilson’s disease the first time. A lot of doctors lacked experiences in rare diseases, and are ill informed of its symptoms / dosages of drugs (it can depend on the individual e.g. their weight), although recognition towards Wilson’s disease may have risen since then.

MAIN CONCLUSIONS

• Diagnosis of Wilson’s is very difficult. Lab tests can identify mutations in the affected protein, but it is a heterogenous condition – you might not develop the disease; the condition might be mild or non-existent. Most patients show symptoms in their 20s. It is not economical to screen everyone for Wilson’s.
• Symptoms are quite varied, from severe to asymptomatic. This depends on the person and damage can be minimised by early diagnosis.
• Univar is monopolising Penicillamine and Trientine production & distribution throughout Europe, and has recently increased their prices by 7 times. Currently, there are no alternatives for Wilson’s disease patients, as it is a rare disease, so patients have to wholly depend on Univar.
• Current pills have to be taken 4 capsules a day, morning and night. It is easy to forget to take the pills, and the pills have to be refrigerated.
• Penicillamine is strong and can have side effects. Patients can experience a period of worsening health conditions such as vomiting before they start to get better. It could take more than a year for improvement.
• Trientine is milder and has less side effects, so informed patients sometimes prefer to be treated with Trientine. However, as Trientine became expensive, the NHS policy is to first administer Penicillamine to patients and only change to Trientine when the patient shows intolerance towards Penicillamine.
• Many doctors lack awareness on rare disease, and sometimes can be dismissive. Different patients react differently towards the same treatment. It depends on where the symptoms are occurring, how much copper has already built up, and how much tolerance the patient has. Due to lack of data and experience, administration of drugs can be a trial and error process.
• Often you will be the only patient in your county, and public awareness is extremely low. Lack of employment opportunities.
• As treatment for Wilson’s disease can be rare, it is difficult to see whether the treatment doesn’t conflict with other drugs for other illnesses.
• Patients will be willing to try an alternative, cheaper treatment if it is proven that it works.

We even won a candle from the raffle!

 

Bacteriocins and the Evolution of Spite

Alex Tep

A more technical post. It will be helpful to be familiar with the idea of the ‘selfish gene’ in evolutionary biology before reading this.

Although our treatment involves E. coli – a species of bacteria that naturally occurs in the gut – creating a stable population of engineered bacteria in the gut will be tricky. Even if our E. coli are able to establish themselves in the gut, they must compete with native gut E. coli for the exact same resources. This is a big problem – our engineered bacteria must allocate some resources that could be used for reproduction to producing the copper chelator. Meanwhile, since native E. coli can commit all their resources to reproduction, they will necessarily be better at reproduction than our E. coli, and will gradually become better represented in the gut than the treatment bacteria until our bacteria are totally expunged.

To prevent this from happening, we are arming our bacteria with bacteriocins – proteins that kill other bacteria. This will allow our E. coli to kill its native rivals, preventing them from being outcompeted. But to release this toxin, E. coli cells must burst open suicidally. How could natural selection favour a gene for a trait that harms its bearer? Well, if the recipient is related to the bearer, it will have a greater than average chance of sharing the same gene as the bearer; the closer the relation, the larger this probability. So if an individual sacrifices itself to save the lives of several close relatives (and the copies of the altruistic gene carried by them), although one copy of the gene is lost, several are saved. Over time, this gene, and the trait it encodes, can become stable in the population. But explaining the existence of traits that harm both actor and recipient, like bacteriocins – well, that is more difficult.

Darwin made pioneering successes in explaining adaptation, but he struggled with behaviours that harm the individual performing them. Regarding altruism, traits that reduce an individual’s own reproductive success – or fitness – to raise another’s, he wrote:

 ‘He who was ready to sacrifice his life, as many a savage has been, rather than betray his comrades, would often leave no offspring to inherit his noble nature.’ (Darwin, 1871)

His solution to this paradox was to look at the effect of altruism on the group of which the individual is a part.

‘A tribe including many members who… were always ready to give aid to each other and sacrifice themselves for the common good, would be most victorious over most other tribes; and this would be natural selection.’ (Darwin, 1871)

But this group-selectionist view of altruism fell out of favour in the twentieth century: altruistic groups were too vulnerable to subversion by selfish cheaters. A free-riding individual joining the tribe that received this aid from other members but refused to help them in return would increase in fitness. And their offspring, who inherit their selfish nature, would increase in frequency at the expense of their comrades’, until there are no altruistic individuals left.

In a series of influential papers, W D Hamilton demonstrated that there is no need to invoke group selection to explain altruistic traits (Hamilton, 1964). When natural selection acts on genes rather than groups, it is possible for a gene for an altruistic trait to increase in frequency if the altruism is directed at individuals that share this gene. More formally, this can be expressed as Hamilton’s rule, which states that selection can act on and favour genes for altruistic traits when:

rB > C

(1)

or when the benefit to the recipient of the action (B), adjusted by its relatedness to the actor (r) – loosely, the likelihood that the recipient shares such a gene, outweighs the cost to the actor (C). This emphasised the value of considering the fitness of relatives as well as that of the focal individual– inclusive fitness – when thinking about social traits.

But as well as explaining altruism, Hamilton’s rule carries a darker suggestion: that it is possible to satisfy the inequality rB > C or, more generally rB  – C > 0, when C is positive and B is negative. Where altruism is helping another at personal cost, spiteful traits harm both actor and recipient (Hamilton, 1970). Explaining how natural selection could possibly favour such mutually destructive qualities has been more problematic.

Firstly, the keen-eyed will notice that in order for Hamilton’s rule to be satisfied in cases of spite, if B is negative and C is positive, r must also be negative:

(2)

But how is it possible to be negatively related to someone? True, if we think of relatedness as the chance of sharing a gene, r cannot be negative because it is a probability. But Hamilton’s rule is much more useful to us if we think of relatedness as a regression coefficient (which can take negative values) of the recipient’s genes on the actor’s (Hamilton, 1963). In his geometric view of relatedness, Grafen (Grafen, 1985) emphasises the value of considering the genetical composition of the population: if the recipient of a social trait carries the actor’s genes at a frequency p, that is greater than the genes’ frequency in the population , increased reproductive success of the recipient means an increased frequency of the actor’s genes in the population, and thus a gain in the actor’s inclusive fitness (RB > 0 (Eqn. 1)). If the recipient carries the actor’s genes at a frequency lower than their frequency in the population, an increase in the recipient’s reproductive success means a reduction in the frequency of the actor’s genes in the population, and thus a loss in inclusive fitness for the actor (RB < 0 (Eqn. 1)). And if p = , relatedness is zero. This is important because changing the value of  alone can change the outcome of inclusive fitness for the actor. So negative relatedness simply means that the recipient’s relatedness to the actor is less than its expected relatedness to a random member of the population.

Second, though negative relatedness is possible, we can normally only expect relatedness between an actor and its social partner to be only slightly negative – so slight as to be considered a trivial force in Hamilton’s rule. An individual living in a population of size N is related to itself, a fraction  of the population, by amount 1, and to the rest of the population, , by amount R. As above, relatedness to the population as a whole must be zero, so:

Rearranging gives us the average relatedness between the actor and its social partners:

(3)

If the population is of any substantial size, N will be large enough to make R only weakly negative. For this reason, Hamilton himself did not place much importance on spite because of its restrictive requirements – populations would have to be tiny for R to be negative enough to have any effect – in his words, spite is merely the ‘final infection that kills falling twigs off the evolutionary tree’ rather than an significant biological phenomenon (Hamilton, 1996).

Indeed examples of spite have been rare, and many of those that have been found actually turned out to be examples of selfishness – actions that harm the recipient to increase the actor’s inclusive fitness. For example, that herring gulls commit siblicide on chicks at neighbouring nests has been cited as an example of spite (Pierotti, 1980), but since this action reduces competition for themselves and their offspring in the future, this is really selfishness (Foster, Wenseleers and Ratnieks, 2001). As West and Gardner put it: ‘[w]hat matters for natural selection are fitness consequences over the entire lifetime and not some arbitrary period’ (West and Gardner, 2010).

But while populations may be large, social interactions do not always occur on a global scale. When looking at a much smaller, more local social arena – for example in the presence of local competition, R can be sufficiently negative to allow for spiteful traits.

Further, the magnitude of R can be boosted if the actor is able to discriminate between closer and more distant relatives. How could this ability arise? Suppose there arose a gene that conferred some conspicuous phenotype, say, for a green beard, and the ability to recognise such a trait and treat bearers of this trait preferentially. This greenbeard gene would increase in frequency in the population by directing altruism towards carrier-relatives and spite towards non-relatives.

E O Wilson has disputed the necessity of negative relatedness altogether for the evolution of spite. He suggested that spite towards non-negatively related individuals could be favoured if the action helped a relative to a more-than-compensatory degree (Wilson, 1976). A modification to Hamilton’s rule can incorporate a third-party relative:

(4)

where R₁ is the relatedness to the recipient, R₂ is the relatedness to the third-party relative and D represents any benefits enjoyed by this relative. Using this model, Wilsonian spite can occur when D > 0 and R₂ > 0, even if R₁ > 0. Contrast this to Hamiltonian spite where R₁ < 0 and R₁B > 0 and D is not necessarily > 0 (Gardner and West, 2004).

Then isn’t spite just altruism to a third-party? Why is it useful to distinguish this from altruism? There are obvious differences between spiteful and altruistic traits from a behavioural perspective, but there are some biologically interesting differences too. We expect a positive linear relationship between fitness and relatedness for altruistic traits – the more closely related the actor is to the recipient, the more likely that the recipient will contain the gene for the trait in question, and the more favourable altruism towards the recipient is.

But with spiteful traits mathematical models predict a domed relationship between fitness and relatedness: when the fraction of spiteful individuals is low,  is low and that of non-relatives,  (Eqn. 3) is high, making R only weakly negative (Eqn. 3) and insufficient to outweigh the cost of the spiteful trait. When the focal group dominates the population, there are too few non-relatives for the harm to the recipient to outweigh the cost to the individual. Kin discrimination is often key in providing negative relatedness for Hamiltonian spite – altruism can evolve without kin discrimination if, for example, dispersal is limited so that relatives stay together.Also, we can expect spite, rather than altruism to be favoured when there is local competition since this is a source of negative relatedness.

Observations on bacteriocin-producing bacteria are consistent with the theory. Bacteriocins are antimicrobial compounds secreted by a wide variety of bacteria (Riley and Wertz, 2002). They are costly to produce (cell lysis is required for bacteriocin release in Escherichia coli, but there is a substantial metabolic cost in all producers) and they kill competing susceptible cells through enzyme inhibition, membrane pore formation, and their nuclease activity, among other mechanisms (Riley and Wertz, 2002); so bacteriocin release can be considered to be a spiteful trait. One might argue that, in non-autolysing cells, the trait is selfish rather than spiteful since the individual gains through loss of competition by killing neighbouring susceptible, but bacteriocins are highly diffusible and an actor is unlikely to benefit from the death of a distant competitor (Inglis et al., 2009). Crucially, bacteriocin-production can be considered a greenbeard phenotype since bacteriocins specifically target non-related individuals while leaving relatives untouched because immunity factors are often linked to the toxin gene (Riley and Wertz, 2002).

Inglis et al. used Pseudomonas aerugnosa and a caterpillar model to demonstrate the importance of relatedness in allowing for spiteful traits (Inglis et al., 2009). In vitro studies competed a bacteriocin-producing strain with a susceptible competitor and the initial frequency of the producing strain was manipulated and its fitness measured as growth rate relative to the susceptible strain. At low and high starting frequencies, producer fitness was low, and fitness was greatest at intermediate starting frequencies (Fig. 1a). In vivo studies in the caterpillar gave comparable results. Infecting populations were manipulated to contain a high (99%), intermediate (50%), or low (1%) frequency of the producing strain relative to the susceptible. As expected, caterpillars inoculated with the intermediate population took significantly longer to die than those treated with the high and low populations (Fig. 1b).

1a.

1b.

FIG. 1 (Inglis et al., 2009)

At high producer frequencies, there are only a few competitors to kill, and thus only a few resources to gain by releasing bacteriocin. The increase in inclusive fitness is insufficient to counterbalance the cost of bacteriocin release. At low producer frequencies R is only weakly negative and the impact on susceptible bacteria is negligible and insufficient to outweigh the costs of bacteriocin production.

Is this an example of Hamiltonian or Wilsonian spite? That there are elements of both here suggests that they are just different ways of looking at the same thing: Wilson just used a different reference population to calculate relatedness so that it was not negative. Indeed, Wilson himself did not draw a distinction between the two, his figure in Sociobiology (Fig. 2) illustrates Hamilton’s view of spite, and more careful inspections of the mathematics of both has demonstrated their synonymy.

FIG. 2 (Wilson, 1976)

Behaviours that harm the individual performing them have long remained a problem for evolutionary theory. But even with Hamilton’s explanation of altruistic behaviour, it has often been assumed that spite cannot be favoured for its restrictive requirements. This was to neglect the importance of local competition and a growing cache of examples exists where kin discrimination, negative relatedness and third-party altruism interact to allow for spiteful behaviour.

REFERENCES

Bourke, A.F.G. (2011) Principles of Social Evolution, 1^st edition, Oxford: Oxford University Press.

Darwin, C. (1859) On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life, 1^st edition, London: John Murray.

Darwin, C. (1871) The descent of man, and selection in relation to sex, London: John Murray.

Davies, N.B., Krebs, J.R. and West, S.A. (2012) An Introduction to Behavioural Ecology, 4^th edition, Oxford: Wiley-Blackwell.

Fisher, R.A. (1930) The Genetical Theory of Natural Selection, Oxford: Clarendon.

Foster, K., Wenseleers, T. and Ratnieks, F. (2001) ‘Spite: Hamilton’s Unproven Theory’, Ann. Zool. Fennici, vol. 38, pp. 229-238.

Gardner, A. and West, S.A. (2004) ‘Spite and the scale of competition’, Journal of Evolutionary Biology, vol. 17, pp. 1195-1203.

Grafen, A. (1985) ‘A geometric view of relatedness’, in Dawkins, R. and Ridley, M. Oxford surveys in evolutionary biology, Oxford: Oxford University Press.

Hamilton, W.D. (1963) ‘The evolution of altruistic behaviour’, American Naturalist, vol. 97, pp. 354-356.

Hamilton, W.D. (1964) ‘The Genetical Evolution of Social Behaviour. I’, Journal of Theoretical Biology, vol. 7, February, pp. 1-16.

Hamilton, W.D. (1970) ‘Selfish and spiteful behaviour in an evolutionary model’, Nature, vol. 228, pp. 1218-1220.

Hamilton, W.D. (1996) Narrow Roads of Geneland Volume 1: Evolution of Social Behaviour, Oxford: Freeman.

Inglis, R.F., Gardner, A., Cornelis, P. and Buckling, A. (2009) ‘Spite and virulence in the bacterium Pseudomonas aeruginosa’, PNAS, vol. 106, no. 14, April, pp. 5703-5707.

Pierotti, R. (1980) ‘Spite and altruism in gulls’, American Naturalist, vol. 115, pp. 290-300.

Riley, M.A. and Wertz, J.E. (2002) ‘Bacteriocins: evolution, ecology, and application.’, Annu. Rev. Microbiol, vol. 56, pp. 117-137.

West, S.A. and Gardner, A. (2010) ‘Altruism, Spite, and Greenbeards’, Science, vol. 327, March, pp. 1341-1344.

Wilson, E.O. (1976) Sociobiology: The New Synthesis, 1^st edition, Cambridge: Belknap Press of Harvard University Press.

 

The evolution of our idea

Part II: Genetic circuitry iterations: To “clock” or not to “clock”

After choosing to focus on Wilson’s disease for our project, we started discussing what our genetic circuit would look like. Sam had already found an article published in “Nature” on September 2015 which described a copper chelator called Copper storage protein 1 (Csp1). The thing that stood out about Csp1 was that each protein molecule is able to chelate 52 copper ions! In our minds, this is a very efficient way of packing copper in a bacterium and we could use the protein as a way to absorb copper from the patient’s body into our synthetic microbe.

Another issue we raised early in the development of the circuitry was where would our bacterium reside. We decided that the bacterium was to act in the gut and more specifically in the small intestine (jejunum and duodenum). This would restrict copper entrance into the body and alleviate the initial symptoms (i.e. copper build up and damage in the liver). E. coli is ideal for the gut since it is naturally found there and because non-pathogenic strains do not harm the host.

The following circuits were initially designed by Sam and me (Andreas)

The 2 circuits proposed initially. Numbers and letters in square brackets are indicators of the steps taken for transcription. They are described with more detail in the text

1st circuit (Left): Sam’s idea was to make expression of the chelator (Csp1) inducible by lactose [1] (lac operator). This would mean that the chelator would be produced as soon as food enters the gut. The chelator is periplasmic and so the periphery of the bacterium would be populated with Csp1. Therefore, all dietary copper trying to enter the bacterium would be absorbed in the periplasm and would not enter the cytoplasm. As all chelator proteins fill up with copper ions, less and less copper is absorbed in the periplasm. Copper concentration would start to rise in the cytoplasm, triggering the copper sensitive activator [2] (CsA). The activator protein would, in turn, bind to its operator (Cso) which would cause transcription (and translation) of the CCDB toxin [3]. As toxin levels rise, the bacterium would die and then excreted with the faeces.

Sam’s first circuit is simple and straightforward. The bacteria can persist and replicate, as long as the CsO is not triggered. This allows the bacterial colony to persist in the gut, something that gives constant copper absorption. Ideally this would abolish the need for taking medicine daily. However, there are some disadvantages. There is no guarantee that the bacteria exiting the gut are dead. Alive bacteria can be excreted as well. This would mean that copper absorbing organisms would be out in the open which is not something we want (healthy individuals might get contaminated with our synthetic bacteria. Also, practically we cannot completely exclude copper ions from the cytoplasm. Some will leak through the chelator and some will be able to enter prior to Csp1 production (transcription and translation take some time – the time between lactose induction and Csp1 deposition in the periplasm is long enough to allow copper intake in the cytoplasm). This would kill the bacteria prior to chelating the necessary amount of copper.

2nd circuit (Right): My idea was to make copper absorption a time restrained action. Csp1 expression would either be constitutive or inducible by lactose [a]. As copper will inevitably enter the bacterial cytoplasm from the moment copper-containing food is in the gut, CsA will be produced and CsO will activate its downstream genes [b]. In this construct the downstream genes are the “clock genes” which were part of a scrapped idea (see the “part I” article) [c]. The molecular clock “records” the time from its expression. After 12 hours the KaiABC clock would activate SasA (mediator) which will in turn phosphorylate a transcription factor called RpaA. RpaA, when activated, would bind to its promoter, activating toxin expression and production [d]. The state (i.e. time) at which the clock is found is inherited by the progeny so the whole bacterial population would be synchronised.

The idea’s merits are that no matter where the bacteria are found, they will be killed after 12 hours. This means that there is minimal chance of out-of-body contamination. However this also means that there is no chance of persistence in the gut since the whole bacterial population dies after 12 hours. This solution would require a daily intake of bacteria in a pill or capsule form. Lastly, the circuit has many parts; it’s quite complicated. Although we could characterise a lot of the genes, we were not sure if we would have the time to prove that they all work together (the molecular clock alone has at least six parts!).

From both of these models useful ideas emerged (e.g. the need for persistence of the colony in the gut was established) which helped to inform further iterations…

Coming up next!,

Part III: Copper, temperature sensing and AND gates.

By Andreas Hadjicharalambous

Meet the team!

 

 

 

Sam Garforth – Biochemistry

Eric LeGresley – Biochemistry

Hannah Webb – Biochemistry

Andreas Hadjicharalambous – Biochemistry

Rosie Brady – Biochemistry

Julia Davis – Biomedical Sciences

 

Iain Dunn – Engineering

 

Shu Ishida – Engineering

Harris Vince – Engineering

Alex Tep – Biology

The evolution of our idea

Part I: How we chose our project

One of the unique features of the iGEM competition is that the teams are able to research and develop their own ideas. This makes research fascinating and interesting; it makes the project feel yours.

The Oxford  iGEM team was assembled in December 2015. After the Christmas holidays, weekly meetings were scheduled. In the meetings we had discussions of various problems that we might tackle with our project. Initially, we had four ideas. The following is a small description of each idea we didn’t pursue:

1) Resilin is an insect protein used in flight and is known for its elastic properties. Its elastic efficiency (proportion of stored energy lost as heat) is very low and its elastic resilience (recovery from deformation) surpasses that of the most resilient synthetic plastics, like polybutadiene. Transformation of E. coli with the resilin DNA sequence would allow us to express the protein and produce a functional resilin sample. After extracting it, we could characterise the physical traits of the material (Young’s modulus, isotropism, force-extension properties, etc), propose multiple uses of resilin and possibly develop a sample for the product. Uses could include athletic equipment (e.g. better footwear), replacement for parts of  human tissue (e.g. tendons and cartilage) or possible replacement of  synthetic surgical scaffolds for growth of cells within the patient (resilin is biodegradable and does not induce an immune response, something that happens with material that is currently used).

2) Bacteria pass genetic information to their progeny (vertical gene transfer), as well as between cells in the same generation  (horizontal gene transfer). One of the horizontal gene transfer methods that bacteria use is called conjugation. They do this via cell-to-cell contact using a structure known as a pilus. The pilus shuttles DNA via a plasmid, a circular piece of DNA. For conjugation a specific plasmid, known as the F plasmid, is used. It contains all the genes necessary to produce the pilus, genes for its replication and miscellaneous genes which might aid the bacterium’s survival (e.g. antibiotic resistance genes). The idea was to exploit the process of conjugation to kill pathogenic bacteria in the body of a patient. Our bacterium would contain 2 plasmids: one containing a protein known as CCDA and a modified F plasmid containing CCDB downstream of an inducible promoter (e.g. Lac promoter). CCDA-CCDB is known as a bacterial toxin-antitoxin system. CCDB is a DNA gyrase inhibitor which inhibits DNA replication and causes defects on chromosome segregation and cell division. CCDA is an inhibitor of CCDB. The fact that CCDA is part of our engineered bacteria means that CCDB would not be active. In contrast, since CCDB is on an F plasmid, it can be transferred via conjugation to any possible pathogenic strains. However, CCDA is not and therefore any recipient would only have the CCDB plasmid. Induction via lactose or IPTG would cause expression of CCDB and kill the pathogens. This could provide an alternative to antibiotics to which bacteria are becoming increasingly resistant.

3) Some cyanobacteria (photosynthesising bacteria) contain internal biological mechanisms which allow the organism  to adapt to the predictable environmental changes of the day/night cycle. This is done using a set of “clock” proteins which control the transcriptional frequencies of specific genes according to the time of day (a set of genes is expressed a dusk and another at dawn). This oscillating mechanism is, in some species like Synechococcus elongatus, quite robust even in vitro (such as the KaiABC “clock” proteins, which have been shown to keep their 12hr oscillations for days ). A group at Harvard University has managed to transplant the “clock” proteins into E. coli, an organism which doesn’t have this kind of mechanism for gene regulation. Furthermore, the University of Chicago iGEM team 2015 has managed to characterise and submit some of the KaiABC proteins to the Registry of Standard Biological Parts. The idea was to give these molecular clocks some functionality (e.g. use hormone producing genes (like insulin) in conjunction with the “clock” to give hormone deficient patients a steady dose of the missing hormone or produce light-oscillating bacteria for street lighting).

Although all of the ideas discussed above are very interesting and have potential many had some constrains. Resilin is currently under study by multiple experienced labs all around the world. Many of the stuff we wanted to test are either already being researched or have just been published.

The toxin-antitoxin idea was interesting but many of the human practices that we wanted to do revolved around the use of the system to tackle antibiotic resistance, something thoroughly covered by last year’s Oxford iGEM team.

After extensive research, we found that the  molecular-clock-E. coli would have taken too much time to produce and characterise. On top of that, we would have to connect the system to a functional gene.

Therefore we settled on Gaurav’s idea to treat Wilson’s disease. The concept of synthetic bacteria to treat a genetic disorder seemed quite interesting and feasible.  We started working on the potential genetic circuitry that we could use to solve this problem and during Easter genetic construct design began.

If you want to learn more about Wilson’s disease, read our previous post on the disease: https://oxfordigem2016.wordpress.com/2016/05/19/what-is-wilsons-disease/

Coming up next: Part II: Iterations that our genetic constructs went through

 By Andreas Hadjicharalambous