Part:BBa_K1744002
araC-PBAD-vcrx028-P1U8-amilCP-kanR
Part Description
This part is a fully autonomous arabinose killswitch with a kanamycin resistance gene (truncated version) and amilCP. The araC gene is driven by its native promoter. It controls the expression of vcrx028 (a toxin) through the promoter PBAD. This promoter is inducible with arabinose and can be repressed with glucose (final concentration of 1 and 5%w/v, respectively). vcrx028 is a toxin gene isolated from pVCR94, a conjugative plasmid discovered in the cholera outbreak of 1994 in a Rwanda refugee camp. Then there is amilCP, a chromoprotein isolated from Acropora millepora that serves as a selection marker. Finally, we have the kanamycin resistance gene which is Aph(3’)-I (aminoglycoside 3’-phosphotransferase) and originate from pOK12. It was selected because the coding sequence had a deletion, making the sequence smaller. This is of particular importance for our project because we use this part for recombineering, which is greatly impaired if the length of the cassette used is bigger than 1.5 kb. This part is designed to be used in a recombineering experiment to make a clean deletion where no scars such as FRT sites are left from the experiment. To do so, we first delete the targeted region with our cassette amplified with homology on both sides of the region to delete and then use a medium with glucose (to repress the toxin) and with kanamycin 50 µg/mL to select the recombinants. Then, using a fusion PCR of both sides of the deletion, the cassette is removed and we can counterselect with arabinose (which induce the toxin and kills cells that did not recombined).To minimise the cassette length and maximise the recombination frequencies we recommend using phosphothiolated primers, 80 bp homologies at least on both sides of the targeted region, only use the part of the cassette PBAD-vcrx028-P1U2-amilCP-KanR and do the recombineering in a cell containing a plasmidic araC expression. Since amilCP’s expression is not strong enough to really be visible in a genomic context, unlike plasmidic context, amilCP protein production may therefore serve as an easy plasmid detection system to eliminate plasmidic background that could occur during recombineering experiments. The system is designed so it is easy to select the good cells with the correct phenotype and speed up the screening process that is often the longest part of the experiment.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1227
Illegal NheI site found at 1630
Illegal NheI site found at 1653 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1166
Illegal BamHI site found at 1613 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1001
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 983
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