Help with site specific chromosomal integration in E. coli ?

[Cy Mallon]

Hi Everyone,

I'm using site specific integration to integrate a gene construct coding for fluorescent protein DsRed2 into the chromosome of E. coli MC1000. Here is the exact protocol I'm using, described in this paper: http://jbioleng.biomedcentral.com/articles/10.1186/1754-1611-7-12

In short, the protocol uses the phi80 integration system to integrate on the chromosome of any E. coli that doesn't harbor the phi80 phage. You need two plasmids to accomplish the integration. The first is a helper plasmid that has two functions. It helps the other plasmid--the integrative plasmid that carries the construct you want to integrate and would normally be a suicide plasmid--to propagate in E. coli. It also codes for an integrase gene that binds to the integrative plasmid and the host's chromosome at the phi80 attachment site and catalyzes the integration by creating nicks in the strands of both plasmid and host DNA. If you're familiar with integration this is probably common knowledge...

My problem is that I can't get the integration to work. Both my helper and integrative plasmid are in my host, E. coli MC1000--I know this because I see the expression of DsRed2 when I grow the cells. However, the integration, which should, at least in part, be performed by the integrase gene on the helper plasmid, just doesn't occur. I'm sure of this because after I cure the strain of the helper plasmid, colonies fail to grow on selective media. This is an indication that expression I was previously seeing was merely due to the propogation of the plasmids in the host, not due to actual integration on the chromosome. (Otherwise, the host would also acquire a certain antibiotic resistance marker on it's chromosome along with my DsRed2 construct.)

I've tried the integration at various temperatures and with antibiotics to try to select both the helper and integrative plasmid, yet with no success.

Does anyone have any tips or tricks in getting site specific chromosomal integration to work? I can provide more details if necessary.

Cheers,

Cy

[Drew]

Hi Cy,

Are you sure that the attB site in your MC1000's genome is intact & empty (i.e., available for an integration event)?

Cheers,
Drew