Vibrio cholerae conjugation - is Magnesium important? - (Sep/25/2006 )

Hi all,

I am trying to make a mutant in Vibrio cholerae. I have disrupted my allele with a tetracycline cassette taken from pACYA184 and ligated it into a suicide vector. However on conjugation of the plasmid containing the disrupted allele I am having trouble getting any colonies. I am using a suicide vector that requires the pir protein to replicate so I am conjugating using E. coli SM10. The conjugation protocol is as follows:

1. 1-ml of each overnight culture (recipient and donor) is collected and washed with an equal volume of PBS.
2. The washed cells are resuspended in 50-ul of PBS and mixed.
3. The cell mixutre is spotted onto an LB plate and incubated at 37 degrees overnight.
4. The spot is scraped off and resuspended in 1-ml PBS.
5. The resuspended conjugative mixture is plated onto minimal media containing 1ug/ml tetracyline.

The E. coli can't grow on minimal media since its an auxotroph whereas, the V. cholerae can grow. The tetracycline should select for V. cholerae carrying the mutated allele.

Can anyone see anything wrong with this protocol, I have this feeling that MgS04 should be added to the wash medium. Is magnesium important in conjugation?

Thanks.

I don't know about its importance in conjugation, but it is an inhibitor of tetracycline activity. Tet and Mg++ don't mix well.