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Team:Warsaw/Stage2
From 2010.igem.org
Safety
The aim of our safety – kill switch project is to design and measure a system that enables maintaining stable amounts of E.coli during the eucaryotic cell 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. MinC protein slowers the division of E.coli cell but it doesn’t destroy it. In this case, it enables safe delivery of our desired protein to the inside of the eucaryotic cells – in case of leaking it doesn’t cause premature bacterial lysis and release of our desired protein. The system is universal – it can be used in all bacterial species forming FtsZ ring during cell division. The safety system we designed and measured consists of the parts specified: Promoter T7 [BBa_I712074] + rbs [B0032 or B0034] + MinC [BBa_J29040] on pSB plasmid, studied in E.coli BL21 strain. We measured our construct in vivo, in the expression system, using strong T7 promoter, IPTG induced. The functionality of our safety element describes optical density of E.coli (OD) and the number of colony forming units/ ml (cfu/ml) in IPTG-induced innoculates BL21 carrying our construct. We performed both dynamic and static measurements. 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.
