Part:BBa_K4849016:Design

MtrA - periplasmic decaheme cytochrome protein

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 286
  
• 1000
  COMPATIBLE 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.