Difference between revisions of "Part:BBa K5189001"
 
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         <img src="https://static.igem.wiki/teams/5189/bba-k5189001/1.png" width="50%" alt="Figure 1: Gel electrophoresis validation of folA nucleic acids">
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             <caption>Figure 1: Gel electrophoresis validation of folA nucleic acids</caption>
 
             <caption>Figure 1: Gel electrophoresis validation of folA nucleic acids</caption>
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         <img src="https://static.igem.wiki/teams/5189/bba-k5189001/2.png" width="60%" alt="Figure 2: Expression of folA protein in BL21(DE3) analyzed by SDS-PAGE">
 
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             <caption>Figure 2: Expression of folA Protein in BL21(DE3) Analyzed by SDS-PAGE</caption>
 
             <caption>Figure 2: Expression of folA Protein in BL21(DE3) Analyzed by SDS-PAGE</caption>

Latest revision as of 05:45, 29 September 2024


folA

folA


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


folA Gene Documentation

folA Gene

Base Pairs: 480bp
Origin: E. coli; synthetic

Properties

The folA gene encodes dihydrofolate reductase (DHFR), one of the enzymes crucial in the folate metabolic pathway.

Usage and Biology

The folA gene encodes dihydrofolate reductase DHFR, which plays a vital role in the folate metabolic pathway. By overexpressing the intrinsic enzymes 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 folA gene was successfully amplified using PCR, yielding bands of 480bp, as expected. The gene was inserted into the pRSFduet-1 vector using NdeI and XhoI digestion. The recombinant plasmid was transformed into E. coli DH5α, and validation was performed through colony PCR and enzyme digestion. The gel electrophoresis results confirmed successful ligation with the expected band sizes.

Figure 1: Gel electrophoresis validation of folA nucleic acids
Figure 1: Gel electrophoresis validation of folA nucleic acids

The pRSFDuet-metF-folA plasmid was transformed into E. coli BL21(DE3) to evaluate the co-expression of the folA gene. Protein expression was induced using IPTG and analyzed via SDS-PAGE. The SDS-PAGE results displayed distinct bands corresponding to the folA protein, particularly under induction at 37°C.

Figure 2: Expression of folA protein in BL21(DE3) analyzed by SDS-PAGE
Figure 2: Expression of folA Protein in BL21(DE3) Analyzed by SDS-PAGE