Allele exchange tips please (S. Oneidensis MR-1) - (Sep/05/2013 )

Hello.

I've been trying to do an allele exchange in Shewanella Oneidensis MR-1 with no luck. This should have been simple and easy but it's proving to be way more challenging than I thought.

I'm trying to replace a nonessential gene with a chloramphenicol cassette (under a promoter that can express in MR-1). I'm using the plasmid pRE118 containing KmR, SacB, pir-dependent origin and the mobilization site. I cloned my insert (500bp upstream homology -FRT-CmR-FRT- 500bp downstream homology) into the pRE118. I then transformed E. coli (pir+) with the plasmid and mated those with S. Oneidensis. The mating+recombination is pretty darn inefficient. I plate about 10^9 recipient cells (on many plates of course) and I get a handful of colonies (between 5 and 30).

So far so good. However, out of all the times I've done this, all of the colonies are Cm and Km resistant but not sucrose sensitive. I assumed a point mutation in SacB. The medium I'm using is LB without NaCl but I've tried regular LB as well. Also, these colonies take a REALLY LONG time to come up. Shewanella normally grows just a tad slower than E. coli but these buggers take 4-5 days to grow. They are pink (as they should be) so I can distinguish them from contaminants (I've also PCR verified that they are indeed Shewanella). After repeating the mating a billion times I got 1 sucrose sensitive colony. A PCR showed that the plasmid is in there (though I'm still waiting on the primers to see if it's in the right place). I grew this out in permissive media and plated to select for the second recombination event. Alas, all I got were SacB mutants. I did this several times and I'm really frustrated it's not working.

I think my plasmid might have misintegrated (I should know by tomorrow if there's not delay with the primer arrival). I thought that the origin might have mutated and the bacteria are actually replicating the plasmid rather than having integrated it but a plasmid prep showed no plasmid DNA. I'll later edit the post and add in whether or not I have a misintegration.

I'll have to replace other genes as well so some tips would be great! Especially when it comes to doing the mating and the initial integration since that took me WAY too long.

Thanks in advance

SacB selection is pretty bad, in my experience. Have you thought about galK (you need a galK knockout strain). This has the huge advantage of being selectable on galactose plates, and then also counter-selectable on 2-deoxygalactose plates. Since it is the same gene, mutations are much less of an issue. I don't know if this would work in Shewanella.

I have PheS but it's on a non-moblizeable plasmid with ColE1 origin. I tried cloning PheS to replace SacB but I guess the plasmid I have is not the original because what should have been a unique cloning site obviously wasn't - unless my enzyme exhibited some tremendous star activity, which shouldn't be a problem according to the company. Unfortunately that was the only commercially available enzyme that had a unique site (according to Addgene) and didn't cut in the middle of PheS.

I'm trying to only replace the coding sequence since I don't know where the promoter/terminator for PheS is. Otherwise I have 1 more option - a dual EcoRI site that the creators of the plasmid used to clone in the SacB cassette

Free yourself from thinking about cut sites (mostly) by using PCR on your vector with restriction sites in the 5' end. You'll have to choose the 5' end sites to avoid other vector and insert sites, but this is usually not difficult. Don't forget the extra bases 5' of your restriction site. Mutant PheS with p-chlorophenylalanine  works, but (in my experience) is quite sensitive to pH of the medium. Make sure your pH is relatively high (7.5+), at least for E. coli.