25 pET-BccI

Protocol tips

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	One Shot BL21(DE3) chemically competent E. coli bacteria (Invitrogen) were transformed, according to the manufacturer’s protocol, with 10 ng of each of the 26 pET-BccI plasmids carrying the BRP gene, 25 pET-BccI plasmids with the CAT gene and 22 pET-BccI plasmids carrying the CSFVRP gene. Two colonies from each transformation reaction were used to inoculate equal numbers of 3 ml LB/kanamycin pre-cultures, which were incubated overnight at 37°C and at 250 rpm. The following day, 15 ml cultures (LB/kanamycin) were inoculated with the appropriate volume from their corresponding pre-cultures in order to obtain an optical density (OD)600 value 0.1. The main cultures were incubated up to OD600 = 0.6 and then IPTG (Sigma) was added to a final concentration 1 mM.

Protocol tips

One Shot BL21(DE3) chemically competent E. coli bacteria (Invitrogen) were transformed, according to the manufacturer’s protocol, with 10 ng of each of the 26 pET-BccI plasmids carrying the BRP gene, 25 pET-BccI plasmids with the CAT gene and 22 pET-BccI plasmids carrying the CSFVRP gene. Two colonies from each transformation reaction were used to inoculate equal numbers of 3 ml LB/kanamycin pre-cultures, which were incubated overnight at 37°C and at 250 rpm. The following day, 15 ml cultures (LB/kanamycin) were inoculated with the appropriate volume from their corresponding pre-cultures in order to obtain an optical density (OD)600 value 0.1. The main cultures were incubated up to OD600 = 0.6 and then IPTG (Sigma) was added to a final concentration 1 mM.

Publication protocol

One Shot BL21(DE3) chemically competent E. coli bacteria (Invitrogen) were transformed, according to the manufacturer’s protocol, with 10 ng of each of the 26 pET-BccI plasmids carrying the BRP gene, 25 pET-BccI plasmids with the CAT gene and 22 pET-BccI plasmids carrying the CSFVRP gene. Two colonies from each transformation reaction were used to inoculate equal numbers of 3 ml LB/kanamycin pre-cultures, which were incubated overnight at 37°C and at 250 rpm. The following day, 15 ml cultures (LB/kanamycin) were inoculated with the appropriate volume from their corresponding pre-cultures in order to obtain an optical density (OD)600 value 0.1. The main cultures were incubated up to OD600 = 0.6 and then IPTG (Sigma) was added to a final concentration 1 mM. Four hours later, the cultures were centrifuged at 10,000 g, at 4°C for 5 minutes. The cell pellets were dissolved with 6 volumes of 1× SDS loading buffer and subjected to three instant freeze-thaw cycles from 95°C to -80°C and then finally stored at -18°C. Then 5 μl from each lysate were used for Western blot analysis after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Also an equal volume of PiNK Prestained Protein Marker (Nippon Genetics) was used as a molecular weight indicator. The protein bands were electro-transferred to polyvinyl difluoride membranes (Macherey-Nagel) and, after blocking with 2% bovine serum albumin in phosphate buffered saline (PBS), a 1:2000 dilution of a mouse monoclonal anti-polyHistidine-Peroxidase antibody (Sigma) was used for detection of the expressed 6-Histidine (His) tagged proteins, followed by 3,3′-Diaminobenzidine (DAB) staining (500 μg/ml DAB (Sigma), 2 mM NiCl2 (Sigma) and 0.02% H2O2 (Sigma) in PBS).

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