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The re-targeted plasmid was transferred to the E. coli donor strain CA434 by heat-shock and then to C. butyricum W5 by conjugation with the donor CA434 as described in http://onlinelibrary.wiley.com.ezproxyberklee.flo.org/doi/10.1046/j.1365-2958.2002.03134.x/full:
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the presence of restriction activities have been found to prevent the introduction of plasmid vectors. DNA DNA transferred by conjugation may escape the function of the restriction endonucleases existing in the recipient.
It was concluded that strain CD3 most likely contains a single restriction endonuclease activity that cleaves at 5′-CATCG-3′. The enzyme was therefore designated CdiI.
No methylase is currently known that is capable of protecting the recognition sequence of CdiI.
We first assembled a vector backbone, pMTL28, lacking such sites (Fig. 2). This plasmid retains two Sau96I sites, flanking the erm gene. In view of the presence
of an analogous restriction activity in strains CD6 (see below), a further derivative was constructed, pMTL29, in which these sites were removed (Fig. 2).
Having derived basic vector backbones a number of derivative vectors were made by inserting restriction fragments encoding fragments encoding three different clostridial replicons into the unique PvuII sites of pMTL28 or pMTL29. Each of the three plasmids obtained were then further modified by insertion of the oriT region of plasmid RK2. The final plasmids obtained, and the replicons they carried, were pMTL9301 (pCD6), pMTL9401 (pCB102) and pMTL9611 (pIP404). All of the inserted fragments lacked CdiI recognition sites. pCD6 was the best.
The three constructed plasmids, together with pCD35ECoriT, were introduced into the E. coli donor strain CA434 (HB101 carrying R702) and conjugations undertaken using the plate mating procedure:
For conjugation experiments, the E. coli donor strain CA434 (HB101 carrying the IncPβ conjugative plasmid, R702) was first transformed with the plasmid to be mobilized. The strain obtained was then grown overnight in L-broth and a 1 ml aliquot was centrifuged at 5 K for 1 min, the supernatant was decanted and the cells were gently resuspended in 1 ml of sterile phosphate-buffered saline (PBS). Centrifugation was repeated and the harvested cells resuspended in a total volume of between 100 and 200 ml of an overnight culture of C. difficile grown in BHI broth. This mating mix was then spotted onto a well-dried BHI agar plate which was then incubated anaerobically for 7 h. The bacterial growth was harvested by flooding the agar surface with approximately 500 ml of PBS and resuspension of the biomass with a sterile spreader. This procedure was then repeated to ensure good recovery of transconjugants, and the cell suspension was plated onto BHI agar medium. E. coli donors were counter selected for by the inclusion of d-cycloserine (250 µg ml−1), cefoxitin (8 µg ml−1) and trimethroprim (10 µg ml−1) to the medium and the appropriate antibiotic to select for plasmid uptake, i.e. erythromycin or thiamphenicol. Plates were incubated for between 24 and 72 h, or until colonies were apparent.
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