Difference between revisions of "Team:Exeter/Project"

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<p id="pp">Before starting the project 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 talked about 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 a toxic protein would be strongly selected for. The length of time for this to happen was estimated to be quite short and if this was the case could have strong implications for the effectiveness of a kill switch over time. To test this we decided to use the ministat to perform a continuous culture. The ministat was developed in the Dunham lab at the University of Washington (Miller et al), each ministat chamber is fed from its own media container via a peristaltic pump and the culture volume is set by the height of the effluent needle in the chamber. Air is bubbled through flasks of water to hydrate it and then used to agitate the culture. We inoculated chambers with freshly transformed E. coli (BL21 DE3) and took samples to test if the kill switch was still viable.  
 
<p id="pp">Before starting the project 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 talked about 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 a toxic protein would be strongly selected for. The length of time for this to happen was estimated to be quite short and if this was the case could have strong implications for the effectiveness of a kill switch over time. To test this we decided to use the ministat to perform a continuous culture. The ministat was developed in the Dunham lab at the University of Washington (Miller et al), each ministat chamber is fed from its own media container via a peristaltic pump and the culture volume is set by the height of the effluent needle in the chamber. Air is bubbled through flasks of water to hydrate it and then used to agitate the culture. We inoculated chambers with freshly transformed E. coli (BL21 DE3) and took samples to test if the kill switch was still viable.  
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Our kill switches were all under the control of the T7 promoter, which we knew to be leaky. It was highlighted to us by Markus Gershater, chief scientific officer at Synthace Ltd that any leakiness in a system would likely produce sub lethal doses of the toxic proteins accelerating selection for mutants without the kill switch.
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Our kill switches were all under the control of the T7 promoter, which we knew to be leaky. It was highlighted to us by Dr Markus Gershater, chief scientific officer at Synthace Ltd, that any leakiness in a system would likely produce sub lethal doses of the toxic proteins accelerating selection for mutants without the kill switch. He also pointed out that chemical and physical means of bio-containment were cheaper and more effective in industry. To investigate this, the ministat was used in our project to simulate on a small scale how a kill switch might be maintained in a large chemostat culture.
 
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Revision as of 19:12, 28 September 2016