Part:BBa_K864012:Experience

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iGEM Team Uppsala University 2012 The copy number of a cousin of pSB4A15Iq(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 share a identical ori, and it is therefore strongly suggested they will behave similar. Read about pSB4C15 for details. The functionality of the lacIq repression, and possibility to induce transcription by IPTG has been assessed for three common lacI-repressed promoters in pSB4C15Iq. The results demonstrates tight repression with a possibility of induction. These parts are identical, except for the resistance cassette. Copy number measurement by 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. LacI repression Relative fluorescence of RFP with different promters in pSB4C15Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units. To investigate the functionality of lacIq repression and the effect of IPTG induction in the pSB4x15Iq backbones, three common lac-repressed promoters were assembled with red fluorescent protein (RFP) in the pSB4C15 backbone and grown with and without IPTG. Flourescence was measured with a fluorescence activated cell sorter (FACS). Read about pSB4C15Iq for details. 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 pSB4x15Iq series, since they share an identical origin. The IPTG repression data demonstrates that the pSB4C5Iq backbone is capable of tightly repressing promoters on the same plasmid, whether it is present in high or low copy number. It also demonstrates that expression can be induced to significant levels by addition of IPTG. It is very probable that this also applies for the other pSB4x15Iq backbones.

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