Team:Tongji China/Light control

Light Control

Safety

Light-off system

We use the light-off system from 2017 SSTi-SZGD to kill our bacteria after effecting.
It contains two parts.

The switch

One is a light-repressed system containing a single light repressor LexA-VVD, and a ColE promoter containing a SOS operator sequence for LexA binding, regulates the expression of target genes.

Upon light exposure, the light-switchable repressor LEVI is induced to form homodimers, which then bind to the operator sequence and repress the activity of a promoter by blocking the attachment of RNA polymerase to the promoter. Removal of blue light results in gradual dissociation of the dimers and transcription activation. I repressor, which can repress the activity of the R-O12 promoter, is under the control of the LightOff system. Upon light illumination, LEVI homodimerizes, then represses the expression of cI repressor and initiates target gene expression.

The Killer

Another is for the target gene to kill the bacteria, there we use a suicide gene that encodes Supernova, which is a mutant form of well-known KillerRed, the first engineered genetically-encoded photosensitizer. Photosensitizer contains chromophores that generate reactive oxygen species (ROS) upon illumination, and it is commonly used for illumination-induced destruction of targeted cells.

In general, it forms a light switch and a killer to control the lives of our bacteria, it confirms the safety of our whole project.
Actually our bacteria release in the gut environment for several hours, then they can be eliminated by our immune system. Our project uses this method to ensure the bacteria to be dead when releasing in the environment. It can be safe for human and friendly to the environment.

Then we have some experiments to verify the kill-switch's function.
However, the plasmid electrotransfection is not quite successful, maybe because the type of bacteria--Pseudomonas aeruginosa has not been used in this method, the plasmid vatality in the bacteria is lower than Escherichia coli, we are searching for the reasons, like the promotor strength, codon degeneracy and so on. We will solve the problem in future.

References:
[1] Chen X, Liu R, Ma Z, et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Res. 2016;26:854–857. doi: 10.1038/cr.2016.74.
[2] Trewin AJ, Berry BJ, Wei AY, et al. Light-induced oxidant production by fluorescent proteins. Free Radic Biol Med. 2018 Nov 20;128:157-164. doi: 10.1016.