Part:BBa_K1744001:Design
P1U8-amilCP-kanR
Design Notes
The amilCP chromoprotein synthesis could be improved for the blue color to be visible in single copy faster. It has been of great concern through-out all the project and that is why we have put the strongest combination of promoter and RBS we have found to be well characterised. Still, expression in single copy is not so intense. In our case, amilCP can still be useful as a marker for plasmid background detection during recombineering experiment. Using only the kanamycin marker for recombineering, the blue colonies would represent plasmid transformants and not good recombinants. This use is good for us though, since background is a great concern in recombineering techniques, where screening can be long.
Sources
The part was constructed using a kanamycin resistance gene from plasmid pOK12 (available commercially) and amilCP from BBa_K592009. The promoter and RBS P1U8 were synthesized and come from Mutalik et al. (Nature 2013).
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 1514
Illegal NheI site found at 1537 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]