Part:BBa_K864005:Experience

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iGEM Team Uppsala University 2012 The copy number of a sibling of pSB4C15(FRT), the BBa_K864001, has been estimated by three methods all pointing to it being maintained at a consistent low copy number in E coli cells. These parts are identical, except for their resistance cassette, and it is therefore strongly suggested they will behave similar. Read about pSB4C15 for details. Measurment of fluorescence by FACS flow cytometry Measurment of plasmid yields from liquid cultures Visual color development of colonies on plates Flow cytometry Relative fluorescence of red cassette (J04450) in different backbones in E coli MG1655, with and without IPTG induction (0.5 mM). Quadruplicates (+IPTG samples) or triplicates (-IPTG). Fluorescence in arbitrary units, not compareable between +IPTG and -IPTG. Error bars are standard deviation. E coli MG1655 was transformed with plasmids of different backbones with the J04450 standard RFP cassette. Liquid cultures were analysed by fluorescence with a Fluorescence activated cell sorter (FACS), quantitivly measuring the fluorescence of individual cells. Due to the active native lacI repression system in the bacteria, the experiment was performed with and without IPTG for promoter induction. Read about pSB4C15 for other measurements. Conclusions Results of fluorescence measurments, plasmid yield and color development on plates all point to pSB4C15 being a true low copy backbone. It is strongly suggested that this result is also valid for the whole pSB4x15 series, since they only differ in their resistance cassette.

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