Part:BBa_K5136000

CYP199A4 WT-His tag

Biology

CYP199A4 is a NADH-dependent cytochrome P450 monooxygenase from Rhodopseudomonas palustris cytochrome P450, a heme-dependent enzyme that is a versatile bio-oxidation catalyst for C–X (e.g., X = H, N, S) bond oxidations. (1) CYP199A4 can also function as peroxygenase. The engineered CYP199A4 peroxygenases showed good functional group tolerance and preferential O-demethylation at the meta- or para-position, indicating potential O-demethylation of H- and G-type lignin monomers. (1)

Construction

We use pET-28a(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Figure 1 DNA gel electrophoresis of the colony PCR products of BBa_K4907128_pSB1C3.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1412 bp

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907128_pSB1C3.

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (45.8 kDa).

Figure 3 DNA gel electrophoresis of the colony PCR products of BBa_K4907128_pSB1C3.

Reference

1. M. Jovanovic, E. Lawton, J. Schumacher, M. Buck, Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system. Fems Microbiology Letters 356, 201-211 (2014).
2. B. Wang, R. I. Kitney, N. Joly, M. Buck, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2, 508 (2011).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 150
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 831
  Illegal XhoI site found at 1231
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 150
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 150
  Illegal AgeI site found at 691
  
• 1000
  COMPATIBLE WITH RFC[1000]