Part:BBa_K5189001
folA
folA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
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.
| None |