Part:BBa_K4759223

T7-RBS1-petH-RBS2-petF(D21Y)

Design

Redox proteins and redox proteases are linked by RBS2, joining the two proteins together. The original base of the amino acid encoding PetF at position 21 is GAT, we mutated it to TAT, Where the amino acid in ferredoxin is mutated from Asp to Tyr.

Usage and Biology

After obtaining the best redox partners PetH/PetF, we performed alanine scanning on petF to speculate which sites had a greater impact on its electron transport capacity. Finally, we found that after mutations in seven of them, the electron transport effect would change greatly, so we mutated the amino acids of these sites into other 19 amino acids by modeling, and selected 23 of them to get better results. (Site-directed Mutagenesis parts can be search from BBa_K4759053 to BBa_K4759075, and constructed modeling screening for redox partners can be search from BBa_K4759076 to BBa_K4759099)

Fig. 1: Fermentation of 23 mutants and control groups

We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.

Fig. 2: 9 mutants + wild-type + negative control, 50 ml/250 ml system fermentation

By verifying the catalytic ability, we found that the substrate conversion of D68P was higher than that of the wild type in the nine strains with high fluorescence intensity, reaching 89.2%.

Fig. 3: A:Fluorescence intensity of wild type with 23 mutants B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1336
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1336
  Illegal NotI site found at 1109
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1336
  Illegal BglII site found at 1647
  Illegal BamHI site found at 1330
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1336
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1336
  
• 1000
  COMPATIBLE WITH RFC[1000]