Part:BBa_K3837011
QS(Lux system) based Kill switch(MazE,F system)
To prevent the cell we designed from falling off the amphibian skin and proliferating elsewhere, we created a kill switch using the Lux gene and MazE, MazF gene so that the cells can survive only in areas with high cell density.
LuxI produces acyl-homoserine lactone (AHL). AHL can pass through the cell membrane, so the concentration of AHL increases as the number of cells increases, and this AHL and LuxR protein bind to stimulate the AHL and LuxR regulated promoters to promote cl repressor transcription. The generated cl repressor suppresses the cl regulated promoter, preventing transcription of MazF.
MazE,F is a suicide system. MazF produces a stable toxin, and MazE produces an unstable antitoxin. mazF cuts a specific site of mRNA and causes apoptosis. mazE is an antitoxin against mazF, which prevents apoptosis.
In our kill switch, the antitoxin, MazE, is continuously expressed, but the toxin, MazF, is regulated by Lux. When the cell is removed from the skin of the amphibian and placed in a new environment, the concentration of AHL is lowered, the transcription of the cl repressor is suppressed, and the transcription of MazF proceeds. Although MazE is continuously expressed, it is unstable, so if more mazF is produced, the apoptosis process is triggered by toxin.
For continuous expression of LuxI and LuxR, we tried to use promoters J23100 and RBS B0034. However, when J23100 and B0034 were used for LuxI and LuxR, the expression level of LuxR/HSL dimer was too high when there was one cell, so another combination of promoter and RBS was found through experiments. We decided to use J23100, B0034 for LuxR and J23106, B0034 for LuxI.
References
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