All free-living bacteria carry the toxin–antitoxin (TA) systems controlling cell growth and death under stress conditions. Based on the nature and mode of action of antitoxins, TA systems have been classified into six different types[1-3]. The product of the toxin gene is a protein, while the product of the antitoxin gene is either a protein (in Type II, IV, V, and VI TA systems) or a non-coding RNA (in Type I and III TA systems). In Type IV TA system, the toxin protein and the antitoxin protein do not interact with each other and the antitoxin antagonizes toxin activity by stabilizing its targets.CbeA-CbtA is one of the Escherichia coli's TA systems, and the toxin protein, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ.CbeA does not form a complex with CbtA but functions as an antagonist for CbtA toxicity.
The fission of bacteria will be suppressed but the growth of bacteria is normal. CbeA was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro. Besides, CbeA can relieve the toxicity of CbtA. Unlike the mostly investigated type II TA system, in which toxin directly kills bacteria. Type IV TA system stagnates the growth of the bacteria.（Fig.1）
Geneguard system is a system that can control the growth of the engineered bacteria and the expression of targeted proteins according to the environment of the bacteria. In terms of the feature of CbtA-CbeA, we designed our Geneguard system (Fig. 2). We used a low-expression-level promoter to express CbtA constitutively. When using an inducible promoter to express CbeA and other proteins of interest, we want to reach the aim that when no inducer exists, CbtA can only be constitutively expressed. Only when the inducer exists, bacteria can proliferate and express targeted protein.
Figure 2. Geneguard system in Jilin_China 2017
Since the Geneguard is a conceptive system, we cannot directly conduct the experiments to this system. After referring to other's work, we designed a characterization construct by which CbtA and CbeA can be induced by different inducers to detect their functions.
We designed Geneguard system and thought that it is of high value to apply. Biosafety is our best advantage in Geneguard system. We hope to solve the biosafety issues with our Geneguard system to reduce the risks of escape of engineered bacteria. For example, compared with traditional fermentation, one of the biggest problem of using engineered bacteria to ferment is biosafety. It is a real problem that we have to face in actual applications.
According to the results of our human practice and the ideas we got from the brainstorm, we designed a method of application. Read more in the next part.
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