Article I used: http://www.sciencedirect.com/science/article/pii/S0168165611003804
strain: Clostridium butyricum strain W5 16S ribosomal RNA gene, partial sequence
GenBank: DQ831124.1 http://www.ncbi.nlm.nih.gov/nuccore/DQ831124?report=GenBank
The original article: http://www.sciencedirect.com/science/article/pii/S0360319906001170
strain: Clostridium butyricum partial 16S rRNA gene, strain VPI3266
GenBank: AJ458420.1 http://www.ncbi.nlm.nih.gov/nuccore/AJ458420
The strains and plasmids used in this study are C. butyricum W5 (isolated
from activated sludge from a sewage treatment plant in South Australia and identified by 16S rDNA
sequencing (GenBank accession no. DQ831124) and the RapID ANA II system previously ( Wang et al., 2007)).
The strain W5 was cultured in Tryptone Soya Broth (TSB) (CM0129, Columbia, HBA, Oxoid) and on tryptone soya agar (TSA),
and cultivated in an anaerobic jar at 35 °C. The anaerobic gas atmosphere was created by
ANAEROGEN (AN2005, Columbia, HBA, Oxoid) in the anaerobic jar. Escherichia coli strains were cultured
in L-broth and on L-agar at 37 °C. For E. coli carrying the retargeted plasmid, the medium was supplemented with chloramphenicol (12.5 μg ml−1).
The target site was identified and PCR primers (modified IBS, EBS1d and EBS2)
for intron re-targeting were designed using a computer algorithm, the access of which is provided
as part of the TargeTron™ Gene knockout system kit (http://www.sigmaaldrich.com). The targeting
region of the intron was amplified following the instructions of the kit and then inserted into
Hind III/BsrG I-digested pMTL007 linear vector to yield pMTL007: Cbu-hbd-414s, which means the
intron on this plasmid is targeted to insert in the sense orientation after base 414 of the hbd
ORF of C. butyricum. The new targeting region in the plasmid was further confirmed by sequencing.
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/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 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 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.
(pCD6 replicon) was found to transfer at high efficiency, at frequencies ranging from 1.2 × 10−6 to 5.5 × 10−5 per donor cell
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then Transconjugant colonies were re-streaked onto TSA plates supplemented with 250 μg ml−1 cycloserine and 15 μg ml−1 thiamphenicol to select for transformants. Transformant colonies from the re-streak plate were used to inoculate 1 ml of anaerobic TSB supplemented with 7.5 μg ml−1 thiamphenicol, followed by induction with 1 mM IPTG. Cells were then washed in 0.5 ml PBS, re-suspended in 1 ml TSB, and incubated for 3 h to recover. Finally, the integration mixture was spread on TSA plates supplemented with 2.5 μg ml−1 erythromycin to select for intron integration.