Allelic Exchange in Pseudomonas aeruginosa - (Feb/20/2013 )

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

That is a good question and I do hope someone would give us some good feed back. My situation is that the PAO1 gene I want to delete is very small, 642bp in size. I am knocking out 74bp from the middle of the gene using the gateway technique for pseudomonas published my Choi and Schweizer in BMC in 2005.

My question is about the size of the deletion and gene size, how much would it effect an allelic exchange deletion?