Hello,
I want to generate a deletion mutant of Acinetobacter baumannii. I want to use the suicide vector pBIISK. I already have the plasmid containing the up- and downstream region of the gene of interest. However, I am not able to transform A. baumannii. I already tried electroporation with sucrose or 10% glycerol, DNA uptake ("Natural transformation") with and without NaCl, transconjugation (horizontal gene transfer) from E. coli S17-1. I also tried to use motility agar plates for natural transformation, but everything failed.
Is there another protocol for transformation?
Thanks a lot!
Hi Manuela,
You are not alone! We have experienced this on many occasions! At the end of the day, the integration of the plasmid is probably a rare event, so the efficiency is low, which is why you might to optimise several areas.
I have a few questions first; what antibiotic are you using for selection, what are you trying to delete, have you been able to transform using a control replicating plasmid? How big are the up/downstream regions? What protocol are you following (from Beate)? Do you see any growth on the selective media but they are negative for the deletion, or do you not see anything growing on the media?
My understanding is that natural transformation only works when the strain is motile, and ATCC19606 is not motile. So this might be doomed before you start.
When making the competent cells you could use log-phase instead of overnight cultures, and keep them on ice instead of room temp. Try different electroporation parameters.
Decrease the concentration of selective agent.
Use more plasmid.
Use more cells to increase the chance of a rare event.
If you can answer a few of my questions I can then have another think about ideas to optimise.
ciao...paul
Hi Paul,
yes, I followed Beate's protocol. I used different concentrations of kanamycin for selection. However, there was no growth. I changed the growth phase of the culture, I used cuvettes with different gap sizes and different amounts of DNA, changed the temperature and also the incubation time after electroporation. I also tried a protocol with 10% glycerol instead of sucrose. But still no growth.
I agree with you that 19606 is not motile. However, I also tested Gottfried's protocol for natural transformation. Surprisingly, I got several clones containing the plasmid, but there was no recombination event and so the plasmid got lost again.
In the end, I changed the plasmid from pBIISK to pVT77 and selection with sodium tellurite. Transformation worked and also the recombination event took place. For seggregation, I used LB medium without salt and 200 µg/ml AZT. I got the clone in less than one week and also comfirmed it by sequencing the genome.
Thanks for your answer!
Excellent. And I am particularly happy that the Na₂TeO₃ worked. Cool!
Excellent that our plasmid and method is also working nicely in your hands.
On this behind-the-paper piece, I specifically talk about how hard a time we had transforming A. baumannii for our most recent paper. Very low transformation efficiency. We think it might be because of A. baumannii's complex polysaccharide capsule.
https://naturemicrobiologycommunity.nature.com/posts/giving-acinetobacter-baumannii-a-choice-death-by-phage-or-death-by-antibiotic?channel_id=346-behind-the-paper
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