Part:BBa_K4759246

T7-RBS1-petH-RBS2-petF(D21Y)-linker-GFP1-10

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. Generally, the method of determining whether the redox partner is suitable is through tedious steps such as the construction of plasmids, heterologous expression, construction of catalytic systems, and detection of conversion rate after catalysis. Therefore, we wanted to find a convenient way to do a quick screening. We used the fluorescent protein sfGFP to successfully construct a sensor to detect redox partners. sfGFP is a superfolder fluorescent protein that emits green light when irradiated with ultraviolet light. What is special about it is that it can be broken into two parts. We divide sfGFP into sfGFP-1-10 and sfGFP-11, and although these two parts are cut off, there is an interaction force between them, and as long as they are properly folded in space, they will emit light again. Thus, redox proteins are fused to the N-terminus of sfGFP-1-10 and Olep to the C-terminus of sfGFP-11, respectively.

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 BglII site found at 2316
  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]