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•••• UNIPV-Pavia iGEM 2010

This E. coli strain has been successfully used to propagate plasmids with R6K conditional replication origin. BW23474 competent cells have been prepared according to a slightly modified version of the simple CaCl2 method (Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), yielding 10^6 CFU/ug of DNA after bacterial transformation of a pSB*** series vector plated on Cm. Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA. The same transformation efficiency was estimated after bacterial transformation of a R6K control plasmid (BBa_K300008 cut with XbaI-SpeI and self-ligated to generate a Cm-resistant R6K plasmid) plated of Cm, while the control strains (MC1061 and DH5alpha) yielded no colonies as expected because they cannot propagate R6K. Miniprep of the transformed strains yielded a DNA concentration of ~90-100 ng/ul (qualitatively comparable with high copy number plasmids).

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