Trying to get DNA into Shewanella can be difficult (if you are using a strain other than MR-1). Many of us use conjugation from a special E. coli strain to our particular Shewanella strains (we use ANA-3). I recently tried incubating the mating reaction at 37˚C and to my surprise (not really) we got a lot more “transformed” colonies compared to our usual incubation at 30˚C, which is not ideal for E. coli but good for Shewanella. I think the increased efficiency is because E. coli is more metabolically active and able to conjugate with Shewanella a lot better than at 30˚C. So next time you set up a conjugation reaction put one at 37˚C and see if you get more colonies out of the reaction.
Advertisement
July 21, 2011 at 6:56 am
Interesting, I would like to give a try next time.
August 5, 2011 at 7:28 am
I’d like to try that as well – I normally do my matings at 20C and they do work fine.
August 11, 2011 at 5:11 pm
Is this with MR-1? My experience is that you don’t need to go to extreme measures to get DNA into S. oneidensis. We use ANA-3 and our transformation frequencies are around 1 in 10E+7 to 10E+8 for a 6 hour mating. Overnight matings increase the frequency. For Tn mutagenesis this is pretty bad. Anything we can do to increase the frequency the better. Thanks for sharing your experience.
August 16, 2011 at 3:08 pm
@ shewanella – in my case is with frigidimarina,o/n mating.
September 3, 2011 at 5:45 am
Is there a source out there to find which plasmids/origins are compatible with Shewanella? I am new to working with MR-1 and am trying to get up to speed with genetic manipulation.
Thanks for the blog!
September 25, 2011 at 11:23 pm
I was told by an expert Shewanella geneticist that Shewanella would replicate most E. coli plasmids. The problem is getting those plasmids into Shewanella. Electroporation is not easy to do in MR-1. If you find out anything let us all know.
Cheers.