Plasmid_Backbone
pSB4K5

Part:pSB4K5:Experience

Designed by: Reshma Shetty   Group: iGEM06_Bangalore   (2007-02-28)
Revision as of 15:09, 27 September 2012 by Agynna (Talk | contribs) (→‎User Reviews)

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Please enter how you used this part and how it worked out.

Applications of pSB4K5

User Reviews

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User:agynna

iGEM Team Uppsala University 2012

Warning: This is not a low copy backbone. The copy number of a sibling, pSB4C5 plasmid, with the same I50042 ori, has been estimated by three methods and compared to the pSB3C5 (with p15A ori) and the pSB4C15. The results shows strong evidence of BBa_I50042 ori having a significantly higher copy number than specified, comparable to or higher than the p15A ori.

  • Measurement of fluorescence by FACS flow cytometry
  • Measurement 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 I50042 for details and other measurments.

Conclusions

The results demonstrate that pSB4C5 has a significantly higher 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 be expanded to the other pSB4x5 backbones, since they all share the I50042 origin of replication. Casual observations also support this result.

The classic pSB4C5, and most likely the whole pSB4x5 series, are not low copy backbones as specified in the registry. They should not be used as low copy backbones. A possible future use of the pSB4x5 series is as a middle copy backbone that is compatible with the existing pSB3x5 (with p15A ori), something that is certainly useful from a syntetic biology standpoint.

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Reshma Shetty

When miniprepped, pSB4K5-I52002 gives high miniprep yield similar to a high copy plasmid. I also successfully used this plasmid to clone 3 constructs that I could not assemble using either pSB1AC3 or pSB3K5. (I think the lower copy number of the plasmid was key to this construction working.)

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