Part:pSB4C5:Experience

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User Reviews

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•• 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.

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