Info : Transposon vector does not replicate in the anaerobic gram(-) bacteria that I am working on. This bacteria is sensitive to chloramphenicol.
When the plasmid was sent via conjugation, transposition event occured and obtained several transposon mutants. Apparently plasmid does not linger that long with conjugation.
Experiment with Electroporation: colonies underwent several subcultures still held the plasmid (vector specific pcr result is positive and colonies are growing on media with chloramphenicol).
Most of the time chlor resistant colonies die when I transfer them from one patch plate to another. I belive transposition never occurred on those colonies.
My other question is why transposition works via conjugation but electroporation. Thanks.
There might be a few explanations for your results. First of all, conjugation is often a very high frequency event, so you might have hundreds or thousands more recipient cells receiving the donor plasmid through conjugation than you do through electroporation, so the appearance of transposition events could just be a reflection of the population size.
Secondly for at least some transposons there is experimental data that suggests the conjugation process itself stimulates or upregulates transposition.
Thanks Michael J. Benedik , I have found articles show the conjugation stimulates transposition event (for mariner transposon as well). If anyone interested: https://jb.asm.org/content/191/4/1239
https://www.frontiersin.org/articles/10.3389/fmicb.2019.00280/full
Despite the fact that conjugation does stimulate (or more accurately de-represses) transposition, I think the main reason is really about numbers of recipient cells getting a plasmid.
Info: There is no established genome engineering tool for the bacteria I am working with. So everthing is pretty much a mystery to solve.
Michael J. Benedik : It is numbers game thats for sure, other thing is we do not even know the efficiency of transposase promoter.
With Electroporation: Plasmid gets in and I obtained several colonies (over 100) resistant to the drug and positive for vector specific pcr. They were positive even after several subcultures. Then they died in different time points. I believe transposition never occured and died bc of the fact that they lost the plasmid.
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