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