Part:BBa_K1744002:Design
Design 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.
Sources
The part was constructed using araC and PBAD of pBAD30 (commercially available), a RBS selected through rational design, the toxin vcrx028 from the conjugative plasmid pVCR94, P1U8 that are 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 (aminoglycoside3’-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.
References
Mutalik K V, Guimaraes C J, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yu A, Lam C, Rodriguez C, Bennett G, Keasling D J, Endy D & Arkin P A, Quantitative estimation of activity and quality for collections of functional genetic elements, Nature methods, 10(4), 2013.
Datsenko KA, Wanner BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci USA 97, 6640(5), 2000.
Reddy TR, Kelsall EJ, Fevat LMS, Munson SE, Cowley SM, Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs, PLoS ONE 10(5), 2015.
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