Difference between revisions of "Part:BBa K864009:Experience"

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iGEM Team Uppsala University 2012

The copy number of a cousin of pSB4C15Iq, 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 the presence of a lacIq 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

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.

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

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).

Cassettes of the PlaI, PLlacO (BBa_R0011) and PT5lac (BBa_K592008) promoters and the pUCori-lacI were assembled with RFP in the pSB4C15Iq backbone, and transformed into E coli DH5alpha. Single clones of these were inoculated in 2 mL LB media with chloramphenicol (12 µg/mL) and with or without IPTG (0.5 mM). Cultures were grown shaking at 37° C for 18 hours, and were visibly confirmed to have grown at steady state for at least 4 hours.

Relative fluorescence of RFP with different promters in pSB4C15Iq, with and without IPTG induction (0.5 mM). Fluorescence is in arbitrary units.

Samples were equilibrated in PBS solution at 1:160 dilution for 1 hour, and then measured in a BD Biosciences FACSaria II . 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Since the purpose of the experiment was to investigate overall repression functionality and the possibility of induction, the samples of with Plac, PT5lac and PLlacO can be considered a triplicate measurement.

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.