Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Frank Sargent
Date created: 2016-10-10 11:00:00
Date modified: 2016-10-11 07:02:58
Truncated Colicin E9 Lacking Bacteriocin Active Domain
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K1962006_sequence (Version 1) |
Description
Colicins are anti-bacterial proteins produced by some strains of E. coli that typically have three domains: a translocation domain; a receptor binding domain; and a cytotoxic domain. This biobrick encodes a truncated version of Colicin E9 which lacks the C-terminal bacterocin domain, which is this case is a DNase.The part has an engineered multiple cloning site at the 3' end, preceding the RFC[10] suffix and regulation double stop codons, containing the following in-frame restriction sites: BamHI, KpnI, SalI, BglII and NheI. The presence of this multiple cloning site will allow for different toxic domains, or other polypeptides, to be fused at the C-terminus of the truncated colicin and thereby generating a synthetic colicin or novel fusion protein in a rapid and facile manner.
Notes
Compliant with RFC[10].Source
We used the Chimera molecular modelling software to determine the position of the DNase domain of Colicin E9, we then designed a truncated Colicin E9, without this domain. The protein sequence was then back translated into DNA and codon optimised for E. coli K-12 before being synthesised by IDT as a gBlock gene fragment. Using oligonucleotide primers we then amplified the truncated colicin E9 with a multiple cloning site containing the following restriction sites: BamHI, KpnI, SalI, BglII and NheI at the 3' end.| Sequence Annotation | Location | Component / Role(s) |
| Truncated E9 MCS | 1,1356 1357,1386 | feature/cds CDS feature/misc sequence_feature |