Part:pSB4T5:Experience

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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.

pSB4T5 Reshma Shetty

When miniprepped, pSB4T5-I52001 does not give as high of yields as expected for a high copy plasmid likely due to mutations in BBa_I52001 that render it nonfunctional as a high copy origin. I am working to address this issue. However, this plasmid should still work fine (it is just not as convenient to prep).

Aberdeen_Scotland 2009

The transformation was twice unsuccessful. Plasmid rescued contained BBa_I52001.

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