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Team:Warsaw/Stage2
From 2010.igem.org
Kill-switch
Kill-switch system: a safety system that maintains stable amounts of E.coli designed for eucaryotic cells transfection and gene delivery
The aim of our kill switch safety project is to design and measure a system that enables maintaining stable amounts of E.coli during eucaryotic cells transfection.
Its functionality is based on MinC protein – a protein that inhibits bacterial cell division. MinC prevents FtsZ ring formation in the polar region of the cell (see background). MinC protein slowers division of E.coli cells but it doesn’t destroy them. In this case, it enables safe delivery of our desired proteins to the inside of eucaryotic cells – in case of leaking it doesn’t cause premature bacterial lysis and release of our desired proteins. The system is universal – it can be used in all bacterial species forming FtsZ ring during cell division.
We performed both dynamic and static measurements (see results). The dynamic measurement describes OD and number of cfu/ml due to IPTG induction time. The static measurement describes OD and cfu/ml using different IPTG concentrations. We used two different controls: one carrying MinC part only on pSB plasmid, IPTG induced, the second carrying our construct without IPTG induction (our control for leaky expression). During the experiment we used different RBSes (B0032 and B0034) for additional regulation of gene expression to study how much different RBSes would influence the general expression of this system.
Finally, we observed filamental cells formation under the microscope. The observation is consistent with theoretical basis of MinC, which stops cell division even tough bacterium can grow and enlarge its size.
