Team:TU Eindhoven/Safety/Kill Switch

Kill Switch

However the kill switch idea is not really necessary for Click Coli as already states in the Biosafety page. If gene transfer occurs, it still misses the clickable DBCO groups and would therefore miss the protective coating. However our idea is a fundamental tool and can thus be used in other further iGEM project. Therefore it is important for other teams to think about a functional kill switch for their own project when using our tool, because in our case Click Coli not necessarily needs a kill switch system.

However we still though about a kill switch system. This system will be active after the bacteria fulfilled their function. This will limit the time the bacteria is active and in this limited time, there will be less time to adapt or mutate.

How does it work? First, the bacterium is induced with IPTG outside the human body. The plasmid in the bacteria has to be IPTG inducible. The IPTG can turn on an IPTG sensitive promotor and then turns up the lambda suppressor (CI) concentration in the cell which will increase exponentially [1]. A Spo0A gene can be implemented behind a promotor that is sensitive for CI. The toggle will activate Spo0A when it reaches the set concentration of CI.

Timer

An increase of phosphorylation state of Spo0A (Spo0A^P) can cause pulses with an increase in amplitude within every pulse [2]. When reaching a certain concentration of Spo0A^P after a certain time, a Spo0A^P sensitive promotor will initiate the cell death.

Spo0A^P

Controlling Spo0A activation includes a lot of interactions shown in Figure 2. Histidine kinases including KinA, transfer phosphates to Spo0A and autophosphates Spo0A through a phosphorelay consisting of Spo0B and Spo0F [2]. However the total level of Spo0A^P is reduced by phosphates for instance phosphates from the phosphorelay are drained by rap through Spo0F and Spo0E dephosphorylates Spo0A^P [2]. A high level of Spo0A^P can induce sporulation in Bacillus subtilis [2]. But in our case a high level of Spo0AP will be necessary to initiate cell death.

Pulses

The increase in amplitude of pulses is caused by a positive feedback loop [2]. The Spo0A activity pulses activate kinase transcription and this will increase the amplitude of the spo0A pulses and an increase in kinase activity increases de Spo0A activity pulses shown in Figure 3 [2].

Conclusion

This kill switch idea is not yet complete for our project and is still a concept. One issue is that the Phosphorelay gene circuit controlling sporulation initiation is found in Bacillus subtilis, which is a gram positive bacterium and not E. coli which is a gram negative bacterium and which we use for our project. Therefore it is hard to implement this system in E. coli. Another thing is that our plasmids are IPTG induced just like this kill switch, which means that that the timer would start as soon as we introduce IPTG for protein expression.

Bibliography

[1] D. Huang, W.J. Holtz, M.M Maharbiz. Journal of Biological Engineering. 6:9 (2012). doi: 10.1186/1754-1611-6-9

[2] J.H. Levine, M.E. Fontes, J. Dworkin, M.B. Elowitz. Pulsed Feedback Defers Cellular Differentiation. PLoS biology. 10:e1001252 (2012). doi:10.1371/journal.pbio.1001252