Part:BBa_K3381005:Design

Mst-CopC-CBM2a

Assembly Compatibility:

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

  Illegal BamHI site found at 785
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Mst-CopC is known to contain three critical residues for copper binding, one of which is a histidine found at the N-terminus. Therefore, it is important to ensure this residue is preserved within our fusion protein, or binding would likely be significantly impacted. In order to introduce a cut site that would not impede binding, we introduced a modified TEV cut site at the N-terminus such that TEV protease cleaves the amino acid sequence right before the N-terminus histidine of Mst-CopC. Additionally, all cut sites for RFc 10 and NdeI/BamHI (required for cloning into pET 11a) were removed. Then a 5-poly-AT + NdeI prefix and a BamHI + 5-poly-AT suffix was added to facilitate cloning into the pET 11a vector by standard assembly. Lastly, the prefix and suffix for RFc 10 were appended.

Source

Sources of DNA sequences, in order of occurrence in the fusion protein from N' to C': His-tag and TEV linker sequence: Retrieved from His-tag and TEV linker that occurs in bacterial expression vector pMCSG7: https://plasmid.med.harvard.edu/PlasmidRepository/file/sequence/pMCSG7.gb Sequence of Mst-CopC (copper-binding domain) retrieved from Genbank (Methylosinus trichosporium O3B): https://www.ncbi.nlm.nih.gov/protein/ATQ66671 Sequence of the CBM linker domain was back-translated from its amino acid sequence, which was retrieved from the following paper describing a natural occurrence of the linker in a protein with a cellulose-binding domain: https://www.jbc.org/content/293/34/13006.full Sequence of CBM2a (cellulose-binding domain) retrieved from DNA sequence of BBa_K863101 (Cellulose-binding domain of Cellulomonas fimi exoglucanase): https://parts.igem.org/Part:BBa_K863101