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Transposon mutagenesis by electroporation of suicide plasmid

electroporation transposon E. coli

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#1 NPPal



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Posted 30 May 2013 - 03:06 PM

Hello everyone,

I am having a problem that I have worked on for quite some time now. I would like to know if anyone has suggestions that can help.

My aim is to generate transposon mutants in E. coli MG1655 (wildtype). I am using the mini-Tn5 transposon system to do this. I use a plasmid described in << http://www.biomedcen...71-2180/11/38>>, that has the Tn5 transposon and the transposase within it. One of the features of this plasmid is that it has a R6K origin of replication, so that it can replicate only in select strains of E. coli (that express the pir gene). MG1655 doesn't support this ori and therefore, the transposon plasmid acts as a "suicide vector" in this strain. Ideally, when the plasmid goes into E. coli, the transposase should be expressed, which will cut the transposon and paste in the genome and the remaining plasmid gets chewed up/diluted out since it cannot be replicated.

My problem is this: I cannot get any transposon mutants if I directly electroporate the plasmid into MG1655.

I get tons of transposon mutants in this strain if I follow biparental mating with a E. coli donor strain that can replicate and transfer the transposon plasmid. But the problem is that when I screen the transposon mutants, greater than 50% of them contain an intact transposon plasmid that is apparently a "suicide vector". I suspect that parts of the donor strain's chromosome is being transferred to MG1655. Obviously, I cannot use mating to get mutants since a large number of my mutants will contain a plasmid, making screeing a nightmare. By the way, I need at least 100,000 mutant colonies for my work. It will be great if I can get mutants through electroporation since I wouldn't have to deal with conjugation strains and their bag of problems.

Things I have tried with electroporation:
1. Increased post-electroporation incubation upto 24 hours and tried different recovery media (LB, M9, SOC, TB etc)
2. Varied growth temperature (before and after electroporation)
3. Tried a bunch of different E. coli K-12 (including DH5a) & B strains. No luck.
4. Changed the promoter of the transposase to a strong constitutive promoter (T5 for K-12 strains & T7 for BL21(DE3)). Not working.
5. I even acquired a plasmid that has a different transposon system on it. Same situation - No luck with electroporation. Works well with mating but plasmid is not "suicide" in MG1655. I have also tried different mating strains (SM10, S17-1 etc). Same problem.

BTW, positive controls ( plasmids with ori that can replicate in MG1655) work beautifully with electroporation.

There are some papers that created Tn5 mutants in E. coli by electroporating a suicide plasmid. I couldn't find these plasmids in any collection. I have looked into Epicentre's Ez-Tn5 kit but it is very expensive when compared to the volume of work I have on my hands.

I hope I made my problem clear. I had a few theories on why electroporation would not work but after testing them out (points 1-5), I am out of ideas.

My last resort is to purify the transposase and make my own transposome system like Epicentre's, but I am not really fond of protein purification.

If your suggestion(s) work, I promise to give due credit.

Thanks for reading,

- splitting hairs

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