Total RNA was subjected to DNase treatment using Turbo DNase (Ambion, UK) and stored at -80°C. RNA integrity was analyzed visually using denaturing 1.2% agarose gel electrophoresis and quantified using a NanoDrop (Thermo Fisher Scientific, USA). Reverse transcription PCR for C10 proteases was performed using the Superscript III One-step RT-PCR system (Invitrogen, USA). Primers used in RT-PCR reactions are documented in Table 4. Primers EVP4593 were added to a final concentration of 200 nM and 200 ng of total RNA added. As a control for DNA contamination, RT-PCR minus reactions was set up where the control reaction only received primers
after the reverse transcriptase step. Aliquots (20 μl from 25 μl) of all samples were analyzed by standard agarose gel electrophoresis. Induction of Bfgi1 and Bfgi2 excision from the B. fragilis 638R genome B. fragilis 638R was grown overnight and then sub-cultured by a 1 in 50 dilution into fresh broth and grown until late log phase. The culture was then exposed see more to either Mitomycin C (0.2 μg/ml), Tetracycline (0.5 μg/ml) UV light (1 mJ/cm2) then grown for a further 12 hours. Acknowledgements The authors gratefully acknowledge financial support from the following sources: University of Limerick PhD studentship to RFT; Science Foundation Ireland grant 08/RFP/BMT1596 to JCC; PWOT is supported by the (Govt. of Ireland) Dept. Agriculture click here Fisheries and Food FHRI award to the ELDERMET project, MycoClean Mycoplasma Removal Kit and by
CSET (Alimentary Pharmabiotic Centre) and PI awards from Science Foundation Ireland. The B. fragilis 638R genome sequence data were provided by the Pathogen Genome Sequencing group at the Wellcome Trust Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/bf/. Permission of J. Parkhill and S. Patrick to use this data is gratefully acknowledged. References 1. Rajilic-Stojanovic M, Smidt H, de Vos WM: Diversity of the human gastrointestinal tract microbiota revisited. Environ Microbiol 2007, 9:2125–2136.PubMedCrossRef
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