Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Kevin Neil
Date created: 2015-09-13 11:00:00
Date modified: 2015-09-18 12:41:04
P1U8-amilCP-kanR
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1744001_sequence (Version 1) |
Description
This part is a positive selection system for recombineering. It contains a kanamycin resistance gene Aph(3???)-I (aminoglycoside 3???-phosphotransferase, truncated version) with its native promoter as well as amilCP, a deep blue chromoprotein. amilCP gene is under the control of a strong constitutive promoter with a strong RBS to make it more intense than previous versions. The cassette is designed to be a double positive selection marker for recombineering. Once amplified with primer bearing homology to the targeted region, the cassette can be integrated in the genome through lambda red recombination. Once integrated, the colonies should become resistant to kanamycin and become pale blue overtime. However, the blue color is actually faint and really slow to become apparent.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.Source
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 synthesised and come from Mutalik et al. (Nature 2013).| Sequence Annotation | Location | Component / Role(s) |
| aph-3'-I Promoter-aph-3'-I amilCP U8 Promoter P1 | 1,612 613,819 820,1488 1489,1509 1510,1548 | feature/cds CDS feature/promoter promoter CDS feature/cds feature/rbs ribosome_entry_site promoter feature/promoter |