Difference between revisions of "Part:BBa K5189004"
 
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             <caption>Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids</caption>
 
             <caption>Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids</caption>
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         <img src="https://static.igem.wiki/teams/5189/bba-k5189004/2.png" width="60%" alt="Figure 2: Expression of mtdA-fchA proteins in BL21(DE3) analyzed by SDS-PAGE">
 
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             <caption>Figure 2: Expression of mtdA-fchA Proteins in BL21(DE3) Analyzed by SDS-PAGE</caption>
 
             <caption>Figure 2: Expression of mtdA-fchA Proteins in BL21(DE3) Analyzed by SDS-PAGE</caption>

Latest revision as of 05:47, 29 September 2024


fchA

fchA


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4
    Illegal NgoMIV site found at 439
  • 1000
    COMPATIBLE WITH RFC[1000]


fchA Gene Documentation

fchA Gene

Base Pairs: 627bp
Origin: Methylobacterium extorquens; synthetic

Properties

Formyltetrahydrofolate cyclic hydrolase, a key enzyme in the C1 transfer pathway of the methylotrophic bacterium Methylobacterium extorquens, is encoded by the fchA gene. This enzyme plays an essential role in the novel L-5-MTHF-producing pathway.

Usage and Biology

Formyltetrahydrofolate cyclic hydrolase, encoded by the fchA gene, is crucial for the C1 transfer pathway in Methylobacterium extorquens. By overexpressing intrinsic enzymes such as dihydrofolate reductase (DHFR) and methylene-THF dehydrogenase (MTHFR), and introducing genes encoding formate-THF ligase (FTHFL), formyl-THF cyclohydrolase (FTHFC), and MTHFD from the one-carbon metabolic pathway of Methylobacterium extorquens AM1 or Clostridium autoethanogenum, an additional pathway was constructed to produce L-5-MTHF.

Cultivation, Purification, and SDS-PAGE

The mtdA-fchA fragment (1488bp) was successfully amplified using PCR. The fragment was inserted into the pETduet-1 vector by digestion with NdeI and KpnI. The resulting plasmid was transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion. Gel electrophoresis results confirmed successful ligation, as indicated by the expected band sizes.

Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids
Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids

The pETduet-ftfL-mtdA-fchA plasmid was transformed into E. coli BL21(DE3) to evaluate the co-expression of the ftfL, mtdA, and fchA genes. Protein expression was induced using IPTG and analyzed via SDS-PAGE. The SDS-PAGE results displayed distinct bands corresponding to the mtdA-fchA proteins, particularly under induction at 37°C.

Figure 2: Expression of mtdA-fchA proteins in BL21(DE3) analyzed by SDS-PAGE
Figure 2: Expression of mtdA-fchA Proteins in BL21(DE3) Analyzed by SDS-PAGE