Difference between revisions of "Part:BBa K5189002"

Revision as of 15:27, 27 September 2024

ftfL

ftfL

Sequence and Features

ftfL Gene

Base Pairs: 1685bp
Origin: Methylobacterium extorquens; synthetic

Properties

Formate-tetrahydrofolate ligase, a key enzyme of the C1 transfer pathway in the methylotrophic bacterium Methylobacterium extorquens, is encoded by the ftfL gene and plays a crucial role in the novel L-5-MTHF-producing pathway.

Usage and Biology

Formate-tetrahydrofolate ligase, encoded by the ftfL gene, is a key enzyme in the L-5-MTHF-producing pathway. By overexpressing intrinsic enzymes 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 path was constructed to produce L-5-MTHF.

Cultivation, Purification, and SDS-PAGE

The ftfL gene (1685bp) was successfully amplified using PCR. The gene was inserted into the pETduet-1 vector by digestion with BamHI and HindIII. 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 ftfL 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 and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the FtfL protein, particularly under induction at 37°C. Western Blot analysis confirmed the successful expression of all three proteins, demonstrating effective co-expression.

Figure 2: Expression of ftfL Protein in BL21(DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)