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Trying to create deletion mutant. Stuck on last step. - (Aug/08/2013 )

Alright, so I've been having a multitude of problems trying to create a deletion mutant. I've worked through them all and now I'm finally on the last step, with the end in sight and now I have another big problem that just has me stumped. 


So I'm trying to create this mutation in bacteria, Pseudomonas to be exact. I'm going to use homologous recombination to create a mutant. Basically I PCR an upstream + downstream region of the gene I want to delete, and fuse them together with a gentamicin resistance gene in the middle so that the gene is replaced with a gentamicin resistance gene. This construct will then be put in a vector and transformed into E. coli, which will transfer the vector + construct into Pseudomonas so that the homologous recombination can happen. Also I should note the gentamicin resistance gene is derived from another plasmid that I PCRed 


So I have the upstream + gentamicin resistance gene + downstream construct inserted into my vector which I have transformed successfully in to E. coli. When I try to do the homologous recombination, I get no colonies on gentamicin plates. I even tried streaking the E. coli strain with the vector + construct onto gentamicin plates and even this doesn't give me colonies. I checked to make sure the E. coli strain does have the plasmid + construct containing the resistance gene and it does.


I suspected there was something fishy with the gentamicin resistance gene. I tried transforming E. coli with the plasmid from which the gentamicin resistance gene was PCR'ed from and I got colonies that grew on gentamicin. I checked the primers and it is amplifying the right region on the plasmid to get the gentamicin resistance gene.


Right now I have no clue what could be going wrong. Does anyone have any suggestions of what I might be able to do? Thanks.


I'd strongly suggest that your next step is to sequence the plasmid you have made, specifically the gentR gene and promoter.