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Realization of Functional Differentiation in Escherichia coli
Synthetic biology,functional differentiation,cell polarization,PopZ,SpmX,DivIVA,asymmetric cell division,cell death,
|Publication Year :||2019|
In the previous studies, E. coli is defined as a classic example of symmetric cell division. Based on the principle of synthetic biology. we aim to break this balance and realize the asymmetric cell division in E. coli platform. We use the PopZ protein as the organizer, it has the property to self-organize and fold into the higher-level structure at the cell pole. We plan to find a protein which can be recruit by PopZ as our adaptor and then fuse another target with it. By this way, the target will be brought to the cell pole via PopZ recruitment, thus, the molecular asymmetry is built.
In this study, we choose SpmX as our adaptor in the beginning. We cut the RNA polymerase (RNAP) into two subunits and fused them with the SpmX individually. By this way, we hope the activity of RNAP can be re-activated at the pole where the PopZ is localized. Thus, we can realize the transcriptional asymmetry. In this experiment, we choose the DivIVA protein as our reporter, the gene expression of which is driven by RNAP. In the previous study, DivIVA is a curvature sensitive protein, which has the property to accumulate and oligomerize at both endpoints in the rod-shaped bacteria such as E. coli, it should be symmetric distribution. To confirm our assumption is correct, we fuse the DivIVA with the superfolder GFP and try to drive its expression by PopZ/SpmX system. The results indicate that we success to break the symmetric distribution of DivIVA. The transcriptional asymmetry can be built by this system.
However, if we want to build the functional asymmetry, this system is still not robust enough. Afterward, SpmX is indicated that it has the property to self-organized. It may cause the activity of RNAP is activated by random. To prevent this situation, we decide to replace one of the SpmX to another adaptor, CpdR. After adjusting, the new NSCC/PopZ system shows the lower noise signal, and the strength of unipolarity has been optimized.
To realize the functional differentiation, we use the beta-lactamase (AmpR) fused with DivIVA as our reporter. We hope that we can build the asymmetric gene expression of the reporter by NSCC/PopZ system. We assume that one of the daughter cells can inherit beta-lactamase by cell division, which has a higher survivability in the condition with Ampicillin. After a series of experiment, we find that both of the daughter cells will die after treating with Ampicillin. However, the cell which inherits the beta-lactamase can survive for a longer time. To some degree, this result confirms that we have realized the functional differentiation in the E. coli platform.
|Appears in Collections:||分子與細胞生物學研究所|
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