Part:BBa_K5136023

OleTJE

Biology

OleT_JE from Jeotgalicoccus sp. ATCC 8456 is the first identified P450 fatty acid decarboxylase, which efficiently catalyzes a single-step decarboxylation of FFAs to form α-olefins by consuming H₂O₂ (as sole oxygen and electron donor) stoichiometrically (1).
It can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2).

Usage and design

OleT_JE can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2).

Construction

We use pET-28b(+) 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 Gene circuit of His tag-OLET_JE.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1521 bp.

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136023_pET-28b(+).

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 (about 48.4 kDa).

Figure 3 SDS-PAGE analysis of His tag-OleT_JE protein.

Reference

1. Jiang, Y., Li, Z., Zheng, S. et al. Establishing an enzyme cascade for one-pot production of α-olefins from low-cost triglycerides and oils without exogenous H₂O₂ addition. Biotechnol Biofuels 13, 52 (2020).
2. Shen Kui-zhong, Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 715
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 715
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 715
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 715
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 715
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 667