I'm currently working on efflux pump inhibitors and I would like to generate some Klebsiella pneumoniae mutants in my clinical isolates. However my expertise level is minimal. Could you give me any advice on what knockout strategy I should use for Klebsiella pneumoniae? Or indicate laboratories where I could learn that.
Kind Regards,
Joao
Hey there Joao,
I have used the lambda-red system extensively in Salmonella typhimurium and it works very well in that organism. Attached is a link to a paper where researchers use the lambda-red system in Klebsiella pneumoniae to make a capsule knockout mutant. This is a great way to go in my experience as you can have a verified mutant in hand 2 days after you get primers.
The generalized strategy for linear transformation of a drug cassette is as follows:
1 design a primer that has 40-50 bases of homology to the upstream end of your KO -target gene followed by ~25 bases of homology to a antibiotic resistance gene of choice (make sure you include the promoter) and (make sure you check your backgound and the lambda-red plasmid for their drug resistances ... you need to uses a different one for the linear transformation)
2 design the corresponding reverse primer with 40-50 bases of homology to the downstream end of your KO -target gene followed by ~25 bases of homology to the down stream end of your drug cassette.
3. do the PCR with the drug cassette as template. Isolate and purify DNA from the PCR. *** this DNA needs to be HIGHLY concentrated and VERY clean (do the alcohol wash step twice) ***
4. prepare your background by moving the lambda-red plasmid in (beware the origin of replication can be temperature sensitive) The plasmid will make proteins that support short-homology crossover events.
5. electroporate your PCR preparation into your desired stain with the lambda-red plasmid, recover for an hour in rich broth then select for the drug in the cassette. You should have colonies the next day.
6. check your transformants with a primer inside the cassette and outside your gene to confirm proper location.
there you have it!
There are some additional considerations
If your gene is in an operon then inserting the cassette can disrupt the expression and/or control of expression of downstream genes. I have linked a paper of my own where I constructed KO mutants that did not disrupt the expression of downstream genes by removing the cassette in an additional step. This "drugless" KO requires primers with FRT sites and a flipase-producing plasmid.
This is good fun! I hope you enjoy it.
http://www.biomedcentral.com/1756-0500/7/13
http://www.ncbi.nlm.nih.gov/pubmed/15466042
Dear Joao Anes,
I am doing the knock out experiment with Klebsiella pneumonia, I would like to ask you one question that whether you succeed with the above method which was suggested by Professor Joseph T Benrod?
I am looking forwarded to receiving your response,
Yours sincerely,
Le Phuong.
I've been trying to cause gene-knockout in K. pneumoniae using the Lambda Red recombination system to no success. We are using FRT cassettes generated with 60 bp homology arms, and are inducing expression of the lambda recombination genes using L-Arabinose but have not found any success. The same kit has been used in my lab for Enterobacter and has worked in the first try.
Is there anything that I'm missing out on? I've spent literally 1.5 years trying to create mutants but to no avail.
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