3 replies to this topic

[gandhi]

Hi all,

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!

[Lynn Shay]

sometimes the counter selection is just not stringent enough...we have the same problem. check out this paper, "Flagellin A is essential for the virulence of Vibrio anguillarum.", they select the second crossover by looking at the morphology of the colony but I never figure out exactly how they did that.

[phage434]

Counterselection is difficult at best. The sacB counterselection works rather poorly, in particular. The major difficulty is that you are working against evolution -- living systems try very hard to survive, and it is easy to knock out a counterselectable marker (or its effects) in many other ways than they way you would like. A good counterselection combines an selection and counterselection in the same marker, which makes the galK knockout good. Selection on galactose medium requires the marker (native galK) to work, while selection on deoxygalactose is lethal (again requiring the galK gene to function).

[gandhi]

Hi all,

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?

Thank's again.