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Safety Training in our team
We had taken several experiments training before this very summer vocation during which we spent to conduct our project.
To ensure the safety of people in our lab as well as the goal of protecting our experiment, firstly we invited our academic adviser Jiong Hong, to give us a guidance to use the laboratory apparatus correctly. Besides, professor Hong also emphasized the potential toxicity of chemical drugs we use, such as Gelred when doing agarose gel electrophoresis, but of course our lab ranks Risk 1, which means it’s very safe for us students as long as we follow the rules in the lab. Moreover, we also invited previous iGEM team members to give us more detailed attention when doing experiments, and it’s helpful very much.
Kill Switch in E.coli
This year our safety work is based on E.coli, since some of our work is on light sensing and imaging system in E.coli. To make a wider application in the future, the biosafety of E.coli containing these parts should be concerned.
Anti-LPS factor (LALF) regulated by lacI
We designed the part based on K541545. We found that R0010 would keep expressing genes downstream constitutively rather than being induced by IPTG. And we've thought that reasons may lie in the limited amount of Lacl (Lactose Inhibitor protein) expressed by E. coil. So we inserted parts which expresses Lacl with a strong promoter before the old one to improve the amount of Lacl and make our lactose operator work. Besides, we also found that Twin arginine signal peptide is an efficient protein export signal sequence of E. coli. Adding this segment to the part makes our killing switch work better.
Anti-LPS factor (LALF) regulated by IPTG
This part is a cell-kill system. Firstly,constitutive promoter could express enough Lacl that is useful for downstream. Secondly, the reporter in the middle of our part wouldn't show signal nor the cell be killed by the part behind unless IPTG or lactose added into the culture medium. No IPTG or lactose: LALF won't express ,G- won't be killed. IPTG or lactose existing: LALF will express ,G- will be dead.
G-: Gram negative bacteria
Principle:
Limulus anti-LPS factor (LALF) binds LPS on anti-gram negative bacterial surface tightly, forming a biofilm. This can prevent nutrient from entering the cell, causing the growth inhibition.
Also, the biofilm can prevent Plasmid spreading to the environment and gene pollution.
Besides, to make LALF across the cell membrane and bind to LPS, we still need an efficient protein export signal sequence of E. coli. leading LALF into extra-cellular environment.
