I have tried to do the gene knockout using the same plasmid in organisms like Vibrio cholera and Shigella flexnerii and failed. It will be very helpful if you can suggest a good protocol for making competent cells and transformation.
It will be very helpful!
Thanks!
Hi, I am using it because I need a conditional knockout using arabinose promoter
Assuming you mean the pBAD33 from this publication: Article Tight Regulation, Modulation, and High-Level Expression by V...
It's not a knockout vector, it's a complementation vector. I believe what they did was:
1. Put an essential gene under control of the arabinose promoter in pBAD33, transformed it into the E. coli strain.
2. Knocked out the gene using Tn10 insertion
3. Showed that these mutants could only grow when supplemented with arabinose.
It's not designed to knock out genes. It will replicate in Shigella and probably Vibrio so you can't even select for chromosomal integration of the plasmid with those. I don't think p15a origins replicate in Acinetobacter, so you might get some chromosomal integration there, but it'll probably be pretty inefficient. There's also no counter selection marker on it to select for double cross-over events which limits its utility a lot.
Alexandra Johnson : Thanks a lot! yes! It was inefficient. We did not do double cross over. It was single cross over.
I tried looking up literature for pbad33 and whether some organisms had the ORI matching it.I could not find it. do you recommend any literature and can I have your email ID if you do not mind. I would like to email about my experiment in detail .
Thanks a lot!
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