Difference between revisions of "Part:BBa K2292006"

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MazEF kill switch

MazEF is a suicide system in Escherichia coli which consists of a stable toxin mazF and labile antitoxin mazE. MazF causes cell death by cleaving mRNA at a specific site. On the other hand, mazE plays a role of antitoxin which can antagonize the toxin mazF.

In general, continuous generation of MazE can prevent the situation of cell death by antagonizing mazF. However, once mazE ceased to be produced, it would be degraded by the protease ClpAP. Hence, the remaining toxin mazF will cause the cell death by cleaving mRNA.

This is a composite of mazEF that is regulated by Arabinose. When arabinose is added, mazEF will be produced. And once arabinose supply is in short, mazE ceased to be produced, it would be degraded by the protease ClpAP.

Figure 1 Mechanism of mazEF kill switch

Figure 2 MazEF function test (OD600 vs. Time)

In the beginning, we add arabinose to the E. coli, and the kill switch start. During the first hour, mazE and mazF are produced. Because of the toxin-antitoxin characteristic, E. coli grows normally in the first hour. After one hour, the mazE and mazF concentrations reach saturation, and then start degrading. Compared with mazF, mazE degrades faster, so mazF cut mRNA for important protein. Finally, E. coli cannot survive. The OD600 value we observe is that it maintains a stable value.

Figure 3 MazEF function test (Plate count method)

Figure 4 MazEF function test (Plate count method)

Following five hours, we test the survival rate every hour (we only see result of negative control and 6 mM Arabinose, because kill switch is independent on concentration of arabinose.) By adding different concentration of arabinose, we found the most appropriate arabinose concentration to induce the suicide system. After five hours, almost all E. coli dead.

Sequence and Features