Difference between revisions of "Part:BBa K5189002"
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| + | <title>ftfL Gene Documentation</title> | ||
| + | </head> | ||
| + | <body> | ||
| + | |||
| + | <!-- Gene Overview Section --> | ||
| + | <h2>ftfL Gene</h2> | ||
| + | <p><strong>Base Pairs:</strong> 1685bp<br> | ||
| + | <strong>Origin:</strong> Methylobacterium extorquens; synthetic</p> | ||
| + | |||
| + | <!-- Properties Section --> | ||
| + | <h3>Properties</h3> | ||
| + | <p>Formate-tetrahydrofolate ligase, a key enzyme of the C1 transfer pathway in the methylotrophic bacterium <em>Methylobacterium extorquens</em>, is encoded by the <em>ftfL</em> gene and plays a crucial role in the novel L-5-MTHF-producing pathway.</p> | ||
| + | |||
| + | <!-- Usage and Biology Section --> | ||
| + | <h3>Usage and Biology</h3> | ||
| + | <p>Formate-tetrahydrofolate ligase, encoded by the <em>ftfL</em> 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 <em>Methylobacterium extorquens</em> AM1 or <em>Clostridium autoethanogenum</em>, an additional path was constructed to produce L-5-MTHF.</p> | ||
| + | |||
| + | <!-- Cultivation and Purification Section --> | ||
| + | <h3>Cultivation, Purification, and SDS-PAGE</h3> | ||
| + | <p>The <em>ftfL</em> 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 <em>E. coli</em> DH5α. Validation was performed using colony PCR and enzyme digestion. Gel electrophoresis results confirmed successful ligation, as indicated by the expected band sizes.</p> | ||
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| + | <!-- Figure 1 --> | ||
| + | <div style="text-align:center;"> | ||
| + | <img src="https://static.igem.wiki/teams/5189/bba-k5189002/1.png" width="30%" alt="Figure 1: Gel electrophoresis validation of ftfL nucleic acids"> | ||
| + | <div style="text-align:center;"> | ||
| + | <caption>Figure 1: Gel electrophoresis validation of ftfL nucleic acids</caption> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <p>The pETduet-ftfL-mtdA-fchA plasmid was transformed into <em>E. coli</em> BL21(DE3) to evaluate the co-expression of the <em>ftfL</em>, <em>mtdA</em>, and <em>fchA</em> 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.</p> | ||
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| + | <!-- Figure 2 --> | ||
| + | <div style="text-align:center;"> | ||
| + | <img src="https://static.igem.wiki/teams/5189/bba-k5189002/2.png" width="60%" alt="Figure 2: Expression of ftfL protein in BL21(DE3) analyzed by SDS-PAGE and Western Blot"> | ||
| + | <div style="text-align:center;"> | ||
| + | <caption>Figure 2: Expression of ftfL Protein in BL21(DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)</caption> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | </body> | ||
| + | </html> | ||
Latest revision as of 05:46, 29 September 2024
ftfL
ftfL
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 901
Illegal NgoMIV site found at 1423
Illegal NgoMIV site found at 1564
Illegal NgoMIV site found at 1623 - 1000COMPATIBLE WITH RFC[1000]
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.
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.
