I'm going to attempt a biparental mating of E. coli S17-1λpir (donor) with a few Enterococcus faecalis isolates (recipient). I've come across some publications that mention the RK2/RP4 transfer machinery (which S17 has chromosomally integrated) can transfer plasmids between gram negative and gram positive organisms... but the only paper I've seen describing a mating protocol of E. coli with Enterococcus just did it to see if it was possible, they didn't attempt to optimize the conditions, and ended up with a pretty low transformation efficiency because of it.
I just wanted to know if anyone has a protocol, publication, or advice on this topic that they could share. It doesn't necessarily have to be from an Enterococcus species either, I imagine a lot of the same principles would apply to mating with other gram positive species.
Hello,
We perform lots of biparental matings between E. coli and Streptomyces gram-positive bacteria. A general protocol can be found here: http://www.openwetware.org/wiki/Streptomyces:Protocols/Conjugation. Not sure how it will relate to entereococcus, but some general things we alter are: 1) Concentration of MgCl2 in the agar plate, generally between 10-50 mM. 2) The agar medium itself - try a range that your gram-positive likes to grow on. 3) Ratio of E. coli to gram positive - you generally want E. coli in excess. 4) Overlay time - generally you overlay the conjugation with a selection antibiotic for the plasmid and another antibiotic (such as nalidixic acid) to prevent the growth of E. coli. When you do this overlay can range from 12-18 h generally, so worth trying multiple time points. 5) You want to to perform the antibiotic overlay (add to 1 mL of MQ and spread over the plate) before you start to see growth of the gram positive strain - if the strain is already growing a lot on the plate you will have difficulty picking out any antibiotic-resistant exconjugants. 6) Once you get antibiotic resistant exconjugants, restreak them to a new plate where your selection antibiotic and nalidixic acid were added to the molten agar. This is done to check for false positives. 7) Before you do anything check how sensitive the gram-positive is to the selection antibiotic - this can help you select the concentration to use.
Hope some of that helps, good luck!
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