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!

Hi everyone,

I am new to the forum and this is my first post.  I've never done this before, but I thought it's worth a shot!

I am designing primers to construct a deletion cassette from Pseudomonas aeruginosa and am wondering what is the recommended amount of bp to leave out?

I have normally left out ~300 bp and it has worked fine in Lb. casei under a different system.  However, I am now wondering if I have constructed cassettes that will not recombine with the chromosome due to the amount of bp left out (larger than 1000bp).  I have constructed my cassettes by amplifying 600-800bp of the 5' and 3' regions of the gene, while leaving out critical sights in the middle. There is a selectable marker inbetween my two inserts.

Any help would be much appreciated!

-gandhi