Difference between revisions of "Team:Exeter/Integrated Practices"

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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 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). We designed the spacer array to target three essential genes polA, rpoC and topA using the deskgen 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>  
 
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 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). We designed the spacer array to target three essential genes polA, rpoC and topA using the deskgen 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>  
  
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#section_2">results</a>).<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>
  
 
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>
 
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>

Revision as of 15:29, 19 October 2016