sacB Counter-Selection to generate double cross-over deletion mutantssacB Allelic Exchange Deletion Mutants Counter-selection Pseudomonas aeruginosa
Posted 20 March 2013 - 12:51 PM
I am making deletion mutants in Pseudomonas aeruginosa by allelic exchange. Basically, I construct a deletion cassette with gentamycin resistance (~700 bp upstream portion of gene----Gentamycin (Gm) Resistance----~700 bp downstream portion of gene) and I clone this into a suicide plasmid with Carbenicillin (Cb) resistance and sacB (Counter-selective measure that inhibits growth in the presence of sucrose in gram-ve bacteria).
I electroporate the plasmid into P. aeruginosa, and select for transformants on LB Gm 30 ug/ml plates. I then patch isolates onto LB Gm 30 ug/ml AND LB Cb 200 ug/ml to differ from single or double crossover. I assume Gm resistant and Cb resistant isolates are Merodiploid, while Gm resistant and Cb sensitive isolates are double cross over events (gene KO).
In my case, I have only seen merodiploid isolates (Gm and Cb resistant), telling me the plasmid is still integrated and I have a WT copy AND a deletion copy still in the chromosome.
To resolve the merodiploid isolates by sacB counter-selection, I have tried growing merodiploids in LB Gm 30 ug/ml and 5% Sucrose, then dilution plating on the same media. I get isolates, however they remain both Gm AND Cb resistant.
Is the counter-selection not tight enough? Also, I believe there are two copies of the gene, so I need to tighten up selection to ensure integration/ recombination at both sights. Any suggestions?
I plan to run a control LB Gm + 5% sucrose plate to determine the level of inhibition caused by sucrose. I will run a colony PCR once this issue gets resolved and will update.
Please, any help is welcomed!
Posted 23 March 2013 - 01:04 PM
Posted 23 March 2013 - 01:20 PM
Posted 10 April 2013 - 05:40 AM
Thank you for the information! I haven't noticed a difference in colony morphology; thank's for the paper. The galK marker sounds like a great system, but I am already invested in using sacB and have some useful updates.
A colony PCR using CDS primers (amplify gene from start to stop codon) revealed that my isolates have two sized amplicons; once of which is the WT sized band. To determine if these isolates resulted from a single cross-over (they contain plasmid backbone with a KO copy and WT copy) or a double cross-over (they contain a clean KO but there is a second WT copy of the gene) I amplified sacB. The isolates do not contain a sacB gene as per colony PCR. This indicates the presence of a second copy of the gene, which I know was probable from the literature.
I am thinking to repeat the process once I remove the Gentamycin marker from the KO copy. I will remake a KO cassette that targets the internal region of the gene (that is now only homologous to the remaining WT copy since this region has been deleted fom the KO copy). Does anyone else have any ideas?