Difference between revisions of "Part:pSB4C5:Experience"

Uppsala_University 2012

iGEM Team Uppsala University 2012

Warning: This is not a low copy backbone. The copy number of pSB4C5 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.

Wisconsin-Madison 2010

pSB4C5-J04450 is not a very low copy vector as described by the registry. It was transformed from the 2010 Distribution, Kit Plate 1, well 3E by the 2010 Wisconsin-Madison team. As it does not contain the high copy origin, I152002, like in the 2009 Distribution, there shouldnt be more than a few copies per cell. It was plasmid prepped over 20 times, and every time it yielded higher concentrations than every other BioBrick vector used by the team. Gel pictures, plasmid prep concentrations, and fluorescent protein production curves all point to a high copy vector.

Reshma Shetty

pSB4C5 is functional. When pSB4C5-I52002 is transformed into TOP10 cells, no colonies are obtained as expected. When pSB4C5-I52002 is cut with NotI, self-ligated (to eliminate ccdB positive selection marker) and transformed, lots of colonies are obtained. Moreover, pSB4C5-I52002 has been used to successfully assemble BioBrick parts. When miniprepped pSB4C5-I52002 gives high miniprep yield similar to a high copy plasmids.

Aberdeen_Scotland 2009

Digestion with EcoRI and PstI worked well and complete digestion was observed. When pSB4C5-I52002 is transformed into XL1-blue cells, colonies are obtained. pSB4C5-I52002 carries the ccdB gene, toxic to wild-type E.coli strains. This plasmid was propagated in our experiments using the Invitrogen strain DB3.1, carrying the gyrA462 allele that confers resistance to this ccdB allele. During the generation of recombinant clones, the ccdB gene was excised and replaced by other BioBricks. For some of these experiments, E.coli strain XL1-Blue was employed. However, other iGEM teams should be aware that XL1-Blue also carries a gyrA allele, explaining our observation that this common E.coli strain was not killed by non-recombinant pSB14C5.