Difference between revisions of "Part:pSB4C5:Experience"

Revision as of 16:46, 29 September 2009

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of pSB4C5

User Reviews

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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.

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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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 <I>Aberdeen_Scotland 2009</I>
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-Mixed digestion results; digestion with EcoRI and PstI worked well in some cases and complete digestion was observed, however, in other cases incomplete digestion was observed. When [[Part:pSB4C5|pSB4C5]]-[[Part:BBa_I52002|I52002]] is transformed into XL1-blue cells, colonies are obtained. This is an unexpected results since this part contains the ccdB 'death' gene.
+Digestion with EcoRI and PstI worked well and complete digestion was observed. When [[Part:pSB4C5|pSB4C5]]-[[Part:BBa_I52002|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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