Part:BBa_K864003:Experience
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Applications of BBa_K864003
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iGEM Team Uppsala University 2012 The copy number of a sibling of pSB4S15, 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.
Flow cytometry 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. The results also show that pSB4C5 has a significantly higer copy number than specified, of the same magnitude as pSB3C5. We are confident to conclude that the copy number regulation of pSB4C5 is broken. This conclusion can also be expanded to the other pSB4x5 backbones, since they share an identical origin of replication. Casual observations also support this result. Thus, we recommend replacing the pSB4S5 for the pSB4S15 for all low copy applications
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