Plasmid_Backbone

Part:BBa_K864000:Experience

Designed by: Erik Lundin   Group: iGEM12_Uppsala_University   (2012-09-23)

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Applications of BBa_K864000

User Reviews

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iGEM Team Uppsala University 2012

The copy number of a sibling of pSB4A15, 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 measurments.

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 pSB4A5 for the pSB4A15 for all low copy applications


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