Welcome to the Wet-Lab GitHub page for iGEM 2021 Bettencourt team! You will find there all the relevant informations and links related to the experimental design and procedures of this project from ideas brainstorming to experimental setups and protocols.
3
stars
0
forks
source link
Deadman & Passcodes kill switches (Chan et al. 2016) #38
One sentence summary:
The authors developed two genetic gene circuits able to kill efficiently E.coli, they can be reprogrammable to for example modify their killing mechanism.
Deadman kill switch
Goal: design a system that can passively activate a biocontainment system (kill switch) for engineered microbes.
How does it work?
It uses a small molecule-binding TF to create a survival state i.e where a specific environmental signal will determine/influences the repression of toxin production. When the environmental signal is lost, the system switches to the death mode (production of toxin killing the cell).
It uses toggle switch architecture (LacI and TetR mutual repression system) to make the kill switch more robust.
Details & Construction
Alteration of the RBS strengths of LacI and TetR in order to favour TetR expression in a single-copy plasmid. aTc - TetR inhibitor- is needed to maintain the survival state, it will be used to block toxin expression (and therefore cell death)
This induced death was increased by combining control of toxin expression and targeting essential protein degradation.
Passcode kill switch
Goal: Construct a hybrid transcription factor design to extend the modularity of the system.
How does it work?
The systems use hybrid LacI-GalR family TFs to expand the range and complexity of environmental signals used to define biocontainment conditions.
it can easily be reprogrammed to restrict cell growth to a new environment
- Details & Construction
Building and generating hybrid TFs
Indentified the boundaries of the environmental sensing modules (ESMs) and DNA recognition modules (DRMs) found in the LacI-GalR family. They then created hybrid TFs that will regulate the promoter by using a small-molecule input defined by the hybrid's ESM.
Long-term circuit stability
Both kill switch systems showed reduced killing efficiency over time. (escaped ones mainly showed inactivating mutations in the toxin genes).
Proposition of the authors adding some additional killing systems into the circuits could reduce the decrease in killing efficiency.
Chan, C., Lee, J., Cameron, D. et al. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol 12, 82–86 (2016).
Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718764/pdf/nihms732216.pdf
One sentence summary: The authors developed two genetic gene circuits able to kill efficiently E.coli, they can be reprogrammable to for example modify their killing mechanism.
Deadman kill switch
Goal: design a system that can passively activate a biocontainment system (kill switch) for engineered microbes.
How does it work? It uses a small molecule-binding TF to create a survival state i.e where a specific environmental signal will determine/influences the repression of toxin production. When the environmental signal is lost, the system switches to the death mode (production of toxin killing the cell). It uses toggle switch architecture (LacI and TetR mutual repression system) to make the kill switch more robust.
Details & Construction Alteration of the RBS strengths of LacI and TetR in order to favour TetR expression in a single-copy plasmid. aTc - TetR inhibitor- is needed to maintain the survival state, it will be used to block toxin expression (and therefore cell death)
Passcode kill switch
Goal: Construct a hybrid transcription factor design to extend the modularity of the system.
How does it work? The systems use hybrid LacI-GalR family TFs to expand the range and complexity of environmental signals used to define biocontainment conditions.
- Details & Construction
Long-term circuit stability
Both kill switch systems showed reduced killing efficiency over time. (escaped ones mainly showed inactivating mutations in the toxin genes). Proposition of the authors adding some additional killing systems into the circuits could reduce the decrease in killing efficiency.