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| − | <p id="pp">We approached our Integrated Human Practices from two perspectives: the traditional, integrating the Human Practices into the lab project; and the unconventional, integrating the human practices into itself. We thought we could make the biggest impact in both Human Practices and the lab if we provided cohesive, reassured arguments for our methods. </p>
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| | <p id="pp">By interviewing academic and industry researchers about their understanding of kill switches, we could analyse whether they are effective biosafety mechanisms and if they are appropriate for use in either industry or academic research.</p> | | <p id="pp">By interviewing academic and industry researchers about their understanding of kill switches, we could analyse whether they are effective biosafety mechanisms and if they are appropriate for use in either industry or academic research.</p> |
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| − | <p id="pp">One of the main factors driving our decision to focus on the use of kill switches in synthetic biology was the nature of their use in iGEM. It seemed that they were often employed as an afterthought and the dedicated kill switch section of the biosafety page on the registry had parts named “oh my god” and “test”. We decided that providing better quantitative data on kill switches would be valuable. We asked individuals from industry and academia about kill switches and how they thought they might be most effectively implemented, we used their input to inform our work inside and outside the lab.<brr><br> | + | <p id="pp">Dr Tom Ellis influenced the design of our kill switches by corroborating our theory that multiple kill switches in one system would reduce the error rate significantly - creating a fail safe, in the same way that broad spectrum antibiotics significantly reduces the chances of failure. We had planned on making an operon of KillerOrange and KillerRed, to both broaden the spectrum of light at which the reactive oxygen species are produced, but also significantly reduce the error rates of one. Dr Ellis argued that multiple kill switches in one system might have <q style="padding-left:0px;padding-right:0px;">less than one in a billion escape rates</q>.</p> |
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| − | We contacted Dr Markus Gershater, chief scientific officer at <i>Synthace Ltd</i>, to ask him what the application of kill switches might be in an industrial setting and what evidence would be satisfactory for their use. Dr Gershater gave the view that kill switches would not be as effective or economical as the physical and chemical bio-containment methods that Synthace currently employ. One of his concerns was increasing the complexity of an industrial strain makes reproducibility and scalability more difficult. Any mis-control of the switch would incur cost as the run of that culture would die. For these reasons Dr Gershater thought that if increased containment is needed, investment in better physical containment is more effective and economical than biological methods. We decided to test how a kill switch might behave in an industrial setting by performing a continuous culture in a ministat (see <a href="https://2016.igem.org/Team:Exeter/Project#culture">here</a> for details). In this way we could test how viable a containment strategy kill switches are when used in industry.<br><br>
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| − | We then contacted Dr Tom Ellis who leads research in synthetic biology and synthetic genome engineering at Imperial College London. His view was that kill switches are not necessarily inherently flawed, but they are a lot more prone to breaking by mutation than other possible mechanisms (e.g. auxotrophies). He suggested that multiple mechanisms could be combined that each have less than 1 in a billion escape rates. This would could give an escape rate of 1 in 10<sup>20</sup>. As a result of this and our own ideas we designed a system that would incorporate both KillerRed, a kill switch already in the registry and KillerOrange, a new part. Both could work together to provide a more effective kill switch and provide a failsafe if one of the kill switches became ineffective due to mutation. Initially we aimed to construct an operon that contained KillerRed and KillerOrange. This was unfeasible with the cloning strategy that we were using as the overhangs that join the ribosome binding site (RBS) to the coding sequence (CDS) would not differentiate between KillerRed and KillerOrange. Constructing KillerRed and KillerOrange on plasmid backbones with different antibiotic resistance markers would allow both to be transformed together. This is a simpler way to test the hypothesis and would be interesting for the future. We also designed a CRISPR based kill switch building on the work of Caliando and Voigt (2015) that would test if having several targets would increase kill switch efficiency. We designed the spacer array to target three essential genes polA, rpoC and topA using the <i>deskgen</i> platform. We selected three protospacers within the CDS of each essential gene. The cleavage sites were designed to be in the first third, the centre third and the final third of the CDS. The spacer array was designed to be carried on the pSB1C3 plasmid under the control of a constituitive promoter (BBa_J23100). Our aim was to investigate whether combining multiple targets would improve efficiency, whether some genes were more effective targets than others and whether targeting multiple protospacers simultaneously was more effective than a single cleavage site. We designed primers to obtain the Cas9 and tracr RNA sequence from BBa_K1218011 provided in the distribution kit. After several attempts transformations remained unsuccessful. The spacer array could also not be produced as a G-block due to the high number of repeating sequences.<br><br>
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| − | We spoke to Prof. Robert Beardmore EPSRC Leadership Fellow in the Mathematical Biosciences at Exeter University. Much of his research has been into antibiotic resistance. We discussed how high selection pressure is applied by prolonged use of antibiotics and how kill switches may be analogous to this. It is clear that cells which develop a mutation that inactivates the kill switch would be strongly selected for. It was estimated that functional loss of the kill switch would occur in a short amount of time as a result, and if this was the case, could have strong implications for kill switch longevity. Because of this discussion we decided to test the effectiveness of kill switches over time in the ministat. We measured the efficiency of KillerRed and KillerOrange over time in a continuous culture and found that within a week the efficiency of the kill switch was reduced to the level of the control. (see <a href="https://2016.igem.org/Team:Exeter/Project#MSE">results</a>).<br><br>
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| − | We then interviewed Professor Richard Titball, leader of the Microbes and Disease research group at Exeter University. We asked him about potential applications of kill switches. He talked about how physical containment methods traditionally used in microbiology may have limitations when applied to vaccines, an area of his research, as they are administered to the population. He thought that if triggered by a specific environmental condition, a kill switch could be an elegant bio-containment solution as it is a system that can be finely tuned. In practice however he was skeptical that kill switches could be made reliable. Interestingly when we discussed the public perception of synthetic DNA and its potential release into the environment, Prof Titball believed that it was an issue that shouldn't be discussed by the scientific community alone, but that the public should be involved in the risk/benefit assessment of the use of genetically modified organisms. This prompted us to find ways to engage the public in order to better their understanding of synthetic biology and include them in the debate. You can see our interview with Prof Titball below.</p>
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| | <p id="pp">Due to time restraints with our project we decided to focus on providing significant analysis on the kill switches individually, however advice from Markus Geshater of Synthase Limited indicating that multiple kill switches in one system would be unfeasible and the different systems would need to be truly orthogonal to be effective. He explained that if in an industrial setting your synthetic system is producing a protein, then having one or multiple kill switches that are reliant on protein production would reduce the efficiency of production of your desired protein. Furthermore these kill switches could potentially be circumvented by the overexpression of a useful enzyme that is being commercially produced. Consequently, we felt assured in dropping the idea for a multiple kill switch system because our kill switches relied heavily on protein production and thus would reduce the efficiency of the product in an industrial setting. Instead of looking at multiple kill switches in the same system we looked to design three distinct kill switches to test and compare the efficiency of enzymatic, metabolic and DNA degrading mechanisms</p> | | <p id="pp">Due to time restraints with our project we decided to focus on providing significant analysis on the kill switches individually, however advice from Markus Geshater of Synthase Limited indicating that multiple kill switches in one system would be unfeasible and the different systems would need to be truly orthogonal to be effective. He explained that if in an industrial setting your synthetic system is producing a protein, then having one or multiple kill switches that are reliant on protein production would reduce the efficiency of production of your desired protein. Furthermore these kill switches could potentially be circumvented by the overexpression of a useful enzyme that is being commercially produced. Consequently, we felt assured in dropping the idea for a multiple kill switch system because our kill switches relied heavily on protein production and thus would reduce the efficiency of the product in an industrial setting. Instead of looking at multiple kill switches in the same system we looked to design three distinct kill switches to test and compare the efficiency of enzymatic, metabolic and DNA degrading mechanisms</p> |
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