Part:BBa_K4849025:Design
MtrC - Outer membrane protein, a decaheme c-type cytochrome
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1793
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1793
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1793
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1793
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 235
Design Notes
We extracted the Gene from Shewanella oneidensis MR-1 (NCBI:txid211586) genomci DNA using Primers that introduced cynogate assmbly system overhangs (refer IGEM Edinburgh 2023 wiki)
MtrC Forward Primer: CAGTgaagacATaATGATGAACGCACAAAAATC
MtrC Reverse Primer: CAGTgaagacATaagcTTACATTTTCACTTTAGTGTGATC
The overhangs were designed to be cut by BbsI - this would allow introduction into the Cynogate Lv0 acceptor vector
MtrC required domestication: It is important to note that MtrC gene had an extra BsaI site which was the restriction sites used in Lv0 -> Lv1 cyanogate assembly. Likewise, the 242bp faint bands visible for MtrC, demonstrating the presence of an extra BsaI site and thus the requirement for domestication.
Domestication involves using primers to make precise modifications to a DNA sequence, in this case, changing a single base pair so the DNA sequence does not have a recognition site for a restriction enzyme. Domestication was done by PCR followed by restriction digestion and ligation. Primers were designed allowing elimination of the restriction site by changing a single base pair. The domestication PCR also introduced new cut sites on the linear DNA allowing digestion and then re-ligation into a plasmid.
Gel Showing MtrC extracted by PCR:
Figure X. Diagnostic digestion on MtrC, showing 242bp band suggesting requirement for domestication.
MtrC Domestication Forward primer: CAGTgaagacATtggacTCGCAAATTTAGAAAT
MtrC Domestication Reverse primer: CAGTgaagaCAGTCCAATCACTGGCATGTC
Source
Shewanella oneidensis MR-1 (NCBI:txid211586)