BBa_K1744002
1
BBa_K1744002
araC-PBAD-vcrx028-P1U8-amilCP-kanR
2015-09-13T11:00:00Z
2015-09-18T12:41:46Z
The part was constructed using araC and PBAD of pBAD30 (commercially available), a synthetic RBS from Mutalik et al. (2013), the toxin vcrx028 from the conjugative plasmid pVCR94, P1U8, a strong constitutive promoter and a strong RBS from Mutalik et al. (Nature 2013), amilCP, a chromoprotein isolated from Acropora millepora that was BioBrick adapted in E.coli by a previous IGEM team (see BBa_K592009) and KanR (Aph(3???)-I (aminoglycoside 3???-phosphotransferase)) from pOK12 (it is a truncated version). In practice, this part was built from two previously submitted parts of our team: BBa_K1744000 and BBa_K1744001.
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 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 target region with our cassette amplified with homology on both sides of the region to delete and then use medium with glucose (to repress the toxin) and with kanamycin 50 ??g/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 with high efficiencies (100% by now)).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-KanR not more and do the recombineering in a cell containing a plasmidic araC expression. Since amilCP???s expression is not strong enough to be visible in a genomic context, amilCP protein production may therefore serve as an easy plasmid detection system to eliminate plasmidic background that could occur during recombineering. 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.
false
false
_2166_
26754
26915
9
false
The killswitch is induced by arabinose. Since arabinose induction is highly dependent on cell metabolism, make sure that the cells will keep their metabolism toward glucose by adding glucose to the medium. To induce the toxin, both in genomic context or in plasmidic context, use arabinose.
false
Frederic Grenier
annotation2455281
1
araC promoter
range2455281
1
1052
1080
annotation2455288
1
RBS
range2455288
1
1234
1245
annotation2455285
1
PBAD promoter
range2455285
1
1177
1204
annotation2455290
1
A > T, was the native stop codon of vcrx028
range2455290
1
1596
1596
annotation2455291
1
promoter-P1
range2455291
1
1624
1658
annotation2455284
1
operator I2 and I1
range2455284
1
1140
1178
annotation2455295
1
kanR
range2455295
1
2560
3171
annotation2455292
1
UTR-U8
range2455292
1
1663
1683
annotation2455289
1
vcrx028
range2455289
1
1246
1623
annotation2455278
1
forward priming site
range2455278
1
1
22
annotation2455294
1
amilCP
range2455294
1
1684
2352
annotation2455279
1
araC
range2455279
1
23
901
annotation2455286
1
T > G
range2455286
1
1205
1205
annotation2455282
1
operator O1
range2455282
1
1088
1109
annotation2455283
1
CAP site
range2455283
1
1131
1144
annotation2455280
1
operator O2
range2455280
1
930
947
annotation2455390
1
kanR-promoter
range2455390
1
2353
2559
annotation2455287
1
TSS
range2455287
1
1213
1213
BBa_K1744002_sequence
1
caattgtctgattcgttaccaattatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtgtctccatacccgtttttttgggctagcgtaggaggcaaaaatgtgggtcatcgagacaaccgacacctttgatgagtggttcgatgctctggatgataccgatagagcaaacgtgctggcttcgatgatggtgctgcgagatagaggccccatgctgtcgaggccatacgcggatactgttaacggttcatcctacagcaacatgaaagagcttcgggtccaaagcaaaggagatcctatcagagcgttctttgcgttcgatccaaagcgtaaggggattcttctctgcgccggtaacaagaccggggacgagaaaaggttttatgaagtaatgatcccaattgcagaccgtgagtttgcggcgcacttggataaattgaagaaggagtgtcgagctcggtacccggggatcctctagtttacagctagctcagtcctaggtattatgctagctttgaaaattaagaggtatatattaatgagtgtgatcgctaaacaaatgacctacaaggtttatatgtcaggcacggtcaatggacactactttgaggtcgaaggcgatggaaaaggtaagccctacgagggggagcagacggtaaagctcactgtcaccaagggcggacctctgccatttgcttgggatattttatcaccacagtgtcagtacggaagcataccattcaccaagtaccctgaagacatccctgactatgtaaagcagtcattcccggagggctatacatgggagaggatcatgaactttgaagatggtgcagtgtgtactgtcagcaatgattccagcatccaaggcaactgtttcatctaccatgtcaagttctctggtttgaactttcctcccaatggacctgtcatgcagaagaagacacagggctgggaacccaacactgagcgtctctttgcacgagatggaatgctgctaggaaacaactttatggctctgaagttagaaggaggcggtcactatttgtgtgaatttaaaactacttacaaggcaaagaagcctgtgaagatgccagggtatcactatgttgaccgcaaactggatgtaaccaatcacaacaaggattacacttcggttgagcagtgtgaaatttccattgcacgcaaacctgtggtcgcctaataaatgagccatattcaacgggaaacgtcttgctcgtcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctattgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccctgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaccttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaa
igem2sbol
1
iGEM to SBOL conversion
Conversion of the iGEM parts registry to SBOL2.1
James Alastair McLaughlin
Chris J. Myers
2017-03-06T15:00:00.000Z