Part:BBa_K4849016:Design
MtrA - periplasmic decaheme cytochrome protein
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 286
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Gel Showing MtrA extracted by PCR:
Figure X. PCR with OneTaq of MtrA, MtrB, and MtrC. A. 1 Kb DNA ladder diagram (New England Biolabs). The ladder is not to scale, and it is provided for reference purposes only.
Gene Sequence (MtrA): ATGAAAAATTGTTTGAAAATGAAAAATTTGTTGCCCGCCTTGACCATTACCATGGCCATGTCCGCCGTGATGGCCTTGGTGGTGACCCCCAATGCCTATGCCTCCAAATGGGATGAAAAAATGACCCCCGAACAAGTGGAAGCCACCTTGGATAAAAAATTTGCCGAAGGCAATTATTCCCCCAAAGGCGCCGATTCCTGTTTGATGTGTCATAAAAAATCCGAAAAAGTGATGGATTTGTTTAAAGGCGTGCATGGCGCCATTGATTCCTCCAAATCCCCCATGGCCGGCTTGCAATGTGAAGCCTGTCATGGCCCCTTGGGCCAACATAATAAAGGCGGCAATGAACCCATGATTACCTTTGGCAAACAATCCACCTTGTCCGCCGATAAACAAAATTCCGTGTGTATGTCCTGTCATCAAGATGATAAACGGATGTCCTGGAATGGCGGCCATCATGATAATGCCGATGTGGCCTGTGCCTCCTGTCATCAAGTGCATGTGGCCAAAGATCCCGTGTTGTCCAAAAATACCGAAATGGAAGTGTGTACCTCCTGTCATACCAAACAAAAAGCCGATATGAATAAACGGTCCTCCCATCCCTTGAAATGGGCCCAAATGACCTGTTCCGATTGTCATAATCCCCATGGCTCCATGACCGATTCCGATTTGAATAAACCCTCCGTGAATGATACCTGTTATTCCTGTCATGCCGAAAAACGGGGCCCCAAATTGTGGGAACATGCCCCCGTGACCGAAAATTGTGTGACCTGTCATAATCCCCATGGCTCCGTGAATGATGGCATGTTGAAAACCCGGGCCCCCCAATTGTGTCAACAATGTCATGCCTCCGATGGCCATGCCTCCAATGCCTATTTGGGCAATACCGGCTTGGGCTCCAATGTGGGCGATAATGCCTTTACCGGCGGCCGGTCCTGTTTGAATTGTCATTCCCAAGTGCATGGCTCCAATCATCCCTCCGGCAAATTGTTGCAACGG
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)
MtrA Forward Primer: CAGTGAAGACataATGAAGAACTGCCTAAAAATG
MtrA Reverse Primer: CAGTgaagacATaagcTTAGCGCTGTAATAGCTTG
G-block (ie, gene with overhangs): CAGTGAAGACataATGAAGAACTGCCTAAAAATGAAAAACCTACTGCCGGCACTTACCATCACAATGGCAATGTCTGCAGTTATGGCATTAGTCGTCACACCAAACGCTTATGCGTCGAAGTGGGATGAGAAAATGACGCCAGAGCAAGTCGAAGCCACCTTAGATAAGAAGTTTGCCGAAGGCAACTACTCCCCTAAAGGCGCCGATTCTTGCTTGATGTGCCATAAGAAATCCGAAAAAGTCATGGACCTTTTCAAAGGTGTCCACGGTGCGATTGACTCCTCTAAGAGTCCAATGGCTGGCCTGCAATGTGAGGCATGCCACGGCCCACTGGGTCAGCACAACAAAGGCGGCAACGAGCCGATGATCACTTTTGGTAAGCAATCAACCTTAAGTGCCGACAAGCAAAACAGCGTATGTATGAGCTGTCACCAAGACGATAAGCGTATGTCTTGGAATGGCGGTCACCATGACAATGCCGATGTTGCTTGTGCTTCTTGTCACCAAGTACACGTCGCAAAAGATCCTGTGTTATCTAAAAACACGGAAATGGAAGTCTGTACTAGCTGCCATACAAAGCAAAAAGCGGATATGAATAAACGCTCAAGTCACCCACTCAAATGGGCACAAATGACCTGTAGCGACTGTCACAATCCCCATGGGAGCATGACAGATTCCGATCTTAACAAGCCTAGCGTGAATGATACCTGTTATTCCTGTCACGCCGAAAAACGCGGCCCAAAACTTTGGGAGCATGCACCCGTCACTGAGAATTGTGTCACTTGCCACAATCCTCACGGTAGTGTGAATGACGGTATGCTGAAAACCCGTGCGCCACAGCTATGTCAGCAATGTCACGCCAGCGATGGCCACGCCAGCAACGCCTACTTAGGTAACACTGGATTAGGTTCAAATGTCGGTGACAATGCCTTTACTGGTGGAAGAAGCTGCTTAAATTGCCATAGTCAGGTTCATGGTTCTAACCATCCATCTGGCAAGCTATTACAGCGCTAAgcttATgtcttcACTG
The overhangs were designed to be cut by BbsI - this would allow introduction into the Cynogate Lv0 acceptor vector
Source
Shewanella oneidensis MR-1 (NCBI:txid211586)
References
1. Ross, Daniel E., et al. "Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1." Applied and environmental microbiology 73.18 (2007): 5797-5808.
2. Breuer, Marian, et al. "Multi-haem cytochromes in Shewanella oneidensis MR-1: structures, functions and opportunities." Journal of The Royal Society Interface 12.102 (2015): 20141117.