Characterization of arabinose induced self- lysis system: BBa_K112000
In iGEM 2020, our original design was to design a part consisting of UV promoter sulAp and T4 lsysis system. However, due to the high leaking expression of SulAp, we are unable to construct that part at last. Instead, we built a part based on arabinose-induced promoter, the part could successfully lysis the bacteria after arabinose induction.
The experiment of the suicide efficiency was investigated using E. coli (strain DH10B), a gradual decline in OD600 value of recombined DH10B (pSB1C3-pBAD-T4) in response to 1mM arabinose could be observed compared to non-transformed ones (control).
Arabinose was considered as a safe food ingredient. Therefore, this year we still choose it to be our inducer of self lysis system. The patient can take a pill of arabinose to start the system and eliminate the engineered bacteria in their intestine. To co-transform the self-lysis system with our OMV-producing vector(pSB1C3 backbone), the antibiotic resistance tag has to be changed(two tags need to be different), therefore, we use pBAD-HisA plasmid as our backbone.
After checking more instructions about araBAD promoter, we found that the arabinose concentration used in 2020(1mM/L≈0.15g/L) is far lower than the manual suggest(2g/L)(M.R. Green, Molrcular cloning a laboratory manual (fourth edition)), we improved our protocol and accelerate the lysis process from 20 hour to 4 hour. (detailed protocol: supplementary material: protocol14)
