While the application prospects of genetically modified Rhamnobacterium are exciting, concerns have been raised about the biosafety of these modified organisms that are accidentally released into the natural environment. Especially given that Rhamnobacterium is a highly adaptable organism, the consequences of their presence in natural ecosystems are unpredictable. Our team is fully aware of this and ensures the safety of all our team members and takes all necessary precautions to ensure that we do not cause any harm to the environment or individuals.

We use Lactobacillus rhamnosus ATCC 7469 to ensure the safety of our project. This microorganism is classified as risk group 1, it also used as vitamin-detecting bacteria and poses a low risk to human safety and the environment.

In order to achieve degradation function, we introduced multiple genes in our chassis strain Lactobacillus rhamnosus. Before constructing the plasmids, we checked the source of the primary gene we wanted to use. These genes are on a whitelist provided by iGEM.

Our projects include microorganisms modified using heterologous gene introduction. In addition, to prevent engineered strains from escaping into the environment and causing contamination, we also chose glucose concentration sensitive promoter and MazF into the suicide system. Usually, the glucose concentrations in the mammalian gut and the external environment, we considered selecting a promoter, PGlu, that is able to respond to changes in glucose concentrations. Follow iGEM rules, when the cells die any bacterial contents to escape and couse potential risks, should be avoid. Therefore, we chose to use the MazF gene. This gene encodes an endonuclease that can cleave RNA at the ACA site and cause microbial death, thereby mediating cell suicide without causing bacterial cell lysis. The promoter PT-αcrp controls the expression of the suicide gene MazF gene, and when glucose concentrations are low, it initiates the expression of the suicide gene to achieve a suicidal effect.

Similar suicide mechanisms were used in the NYMU-Taipei 2017 team project, and we used their results to refine our project.

Figure 1 shows the genetic map of the T4 holin construct pSB1C3-J04450-K112000。

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