Marie Curie Workshop Laboratory Manual Clostridial Gene ...

Day 4 – Prepare to conjugate target organismsYou will generate conjugal donor strains to conjugate Clostridium difficile 630∆Ermand Clostridium sporogenes 10696.Re-transform plasmid DNA into an appropriate E. coli conjugation donor strainsuch as CA434 and inoculate an overnight culture with the transformantsYou will be provided with one (not both) of the following re-targeted pMTL007plasmids which have already been verified by sequencing:pMTL007::Cdi-spo0A-178a (Targets the spo0A gene inClostridium difficile 630∆Erm)pMTL007::Csp-spo0A-249s (Targets the spo0A gene inClostridium sporogenes 10696)The plasmids must be transformed into E. coli CA434 cells to generate conjugaldonor strains which can be used to transfer the re-targeted pMTL007 plasmids into thetarget clostridial strains by conjugation.Each group will only generate one conjugal donor strain, but other groups willgenerate the other conjugal donor strain, so cultures of both required strains will beavailable on Wednesday morning.1. Transform competent E. coli CA434 cells by electroporationTransform the re-targeted pMTL007 plasmid into an aliquot of electro-competent E.coli CA434 cells. Use the electroporation protocol as described on the previous page,except:Add 1µl of the plasmid DNA solution to the aliquot of electro-competent E.coli CA434 cells.Dialysis of the plasmid DNA is not required.2. Inoculate the transformed cells directly into an overnight cultureAfter the one hour recovery period, add the transformation mixture directly into 5 mlof LB broth supplemented with 12.5 µg/ml chloramphenicol. The demonstrators willincubate the culture at 37°C and 200rpm shaking overnight.University of Nottingham, Sept ’06 Page 12