integration vector question - do i need to clone a full gene (May/12/2007 )

Hello Friends,

I am trying to integrate a cfp carrying plasmid into the genomic DNA of a Paenibacillus polymyxa isolate. The plasmid I a musing is supposed to be an integration vector for gram psoitives. Therefore I need to clone a part of a P. polymyxa gene in the mcs which is directly upstream of a promoterless cfp. The vector description reads: "The C-terminal fragment of an ORF from a gram positive bacterium is inserted in frame with the cfp. Transformation back into the gram positive with selection for chloramphenicol resistance recovers integration mutants in which the target gene is fused to cfp and expression is driven from the natural promoter."

My questions are:
1. Does this ORF have to be full gene, coz I have only a partial sequence that has 3 possible ORFs as determined by ORF finder.
2.Do I need to clone the promoter as well or the cfp will be driven by the promoter on the bacterial chromosome which should be ideally located upstream of where the plasmid integrated. Is that what the term 'Natural promoter' in the description mean.

Any views on this will be highly appreciated.

cheers
nifT

1. Does this ORF have to be full gene, coz I have only a partial sequence that has 3 possible ORFs as determined by ORF finder.

No, usually a few hundred bp will do fine. You should be able to determine which of these orfs are real by blast, looking for homologous proteins in other organisms.

2.Do I need to clone the promoter as well or the cfp will be driven by the promoter on the bacterial chromosome which should be ideally located upstream of where the plasmid integrated. Is that what the term 'Natural promoter' in the description mean.

No, definitely not. You want only the coding region of CFP, in frame. The whole idea here is that the CFP is only expressed when your gene of interest is expressed. If you put on the promoter, CFP will always be expressed. This might be a good control.

Are you sure you want to use CFP? It is the hardest GFP like protein to get good detection working. Unless you are doing dual labeling, I'd suggest either GFP or YFP.


How are planning on selecting for modified mutants? This is usually the hardest problem.