Difference between revisions of "Part:BBa K5189003"
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<caption>Figure 2: Expression of mtdA-fchA Protein in BL21(DE3) Analyzed by SDS-PAGE</caption> | <caption>Figure 2: Expression of mtdA-fchA Protein in BL21(DE3) Analyzed by SDS-PAGE</caption> | ||
Latest revision as of 05:47, 29 September 2024
mtdA
mtdA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 834
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 212
Illegal NgoMIV site found at 419 - 1000COMPATIBLE WITH RFC[1000]
mtdA Gene
Base Pairs: 862bp
Origin: Methylobacterium extorquens; synthetic
Properties
The methylenetetrahydrofolate dehydrogenase gene, encoded by the mtdA gene, is a related enzyme of the C1 transfer pathway in the methylotrophic bacterium Methylobacterium extorquens. It is one of the key enzymes in the novel L-5-MTHF-producing pathway.
Usage and Biology
The methylenetetrahydrofolate dehydrogenase gene, encoded by the mtdA gene, is crucial for the C1 transfer pathway in Methylobacterium extorquens. It plays a significant role in producing L-5-MTHF. 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 pathway was constructed upon the native pathway 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.
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.
