Team:UCLouvain/Improve

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 Perspectives

Interspecies conjugation

We’d like to furthermore test our system with different bacteria. If, as we suppose, our kiler bacteria can conjugate with other Gram negative bacteria, our system could kill the antibiotic multi-resistance bacteria usually found in hospital, ... (like Pseudomonas sp., Staphylococcus sp, …) Unfortunately, we don’t had enough time to test it… It will be tested soon after Boston.

Design with complete RP4 plasmid

For better efficiency, we’d like to design the same conjugation/killing system, not with a “light” mobilizable plasmid like we have used, but with a complete RP4 plasmid (about 60k bp) to have a much better conjugation efficiency.

Biosafety aspect

We’d like to add biosafety composant to our system. With a killer plasmid carrying the R6K machinery, our killer plasmid will be dependant of Lambda-Pir. If our plasmid become liberated, it cannot be propagated. We have started such system, with a conjugative strain (pSW23T) carrying the R6K Ori. We have clone in this plasmid, after have change the MCS, our “killer” cassette containing the Cas9 and our killer sgRNA. This plasmid was cloner in S17-1 who have Lambda-Pir. Unfortunately, we don’t had enough time to test it… It will be done soon after Boston.

We also like to add a FRT sequence around, for example, the resistance gene present in our plasmid. With a special enzyme, this antibiotic resistance gene will be “cut” of the plasmid, who will be safier.

Adding a recombination system

As we want to do in a first time (but aborted because of time), we wanted to combine our conjugative “killer” system, with a sequence that could permit our “killer” cassette to recombine into the target bacteria. A such system will be more efficient.

Prevention

As explain above, our system could be tested, in a prevention way, to “clean” the multi-resistant antibiotic strain, whitch is global worldwide problem.

Project Issues

• We encounter few stability problems that we still have to fix.
• Our project lack repetitive experiment.