Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Frederic Grenier
Date created: 2015-09-13 11:00:00
Date modified: 2015-09-18 12:41:46
araC-PBAD-vcrx028-P1U8-amilCP-kanR
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1744002_sequence (Version 1) |
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 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.Notes
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.Source
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.| Sequence Annotation | Location | Component / Role(s) |
| forward priming site araC operator O2 araC promoter operator O1 CAP site operator I2 and I1 PBAD promoter T > G TSS RBS vcrx028 A > T, was the native stop codon of vcrx028 promoter-P1 UTR-U8 amilCP kanR kanR-promoter | 1,22 23,901 930,947 1052,1080 1088,1109 1131,1144 1140,1178 1177,1204 1205,1205 1213,1213 1234,1245 1246,1623 1596,1596 1624,1658 1663,1683 1684,2352 2560,3171 2353,2559 | feature/primer_binding primer_binding_site feature/cds CDS feature/operator operator promoter feature/promoter operator feature/operator non_covalent_binding_site feature/binding feature/operator operator promoter feature/promoter feature/mutation sequence_alteration feature/start start_codon feature/rbs ribosome_entry_site CDS feature/cds sequence_alteration feature/mutation feature/promoter promoter ribosome_entry_site feature/rbs feature/cds CDS feature/cds CDS feature/promoter promoter |