Difference between revisions of "Part:BBa K5189003"

 
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===Usage and Biology===
 
  
 
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===Functional Parameters===
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    <title>mtdA Gene Documentation</title>
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    <!-- Gene Overview Section -->
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    <h2>mtdA Gene</h2>
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    <p><strong>Base Pairs:</strong> 862bp<br>
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    <strong>Origin:</strong> Methylobacterium extorquens; synthetic</p>
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    <h3>Properties</h3>
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    <p>The methylenetetrahydrofolate dehydrogenase gene, encoded by the <em>mtdA</em> gene, is a related enzyme of the C1 transfer pathway in the methylotrophic bacterium <em>Methylobacterium extorquens</em>. It is one of the key enzymes in the novel L-5-MTHF-producing pathway.</p>
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    <h3>Usage and Biology</h3>
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    <p>The methylenetetrahydrofolate dehydrogenase gene, encoded by the <em>mtdA</em> gene, is crucial for the C1 transfer pathway in <em>Methylobacterium extorquens</em>. 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 <em>Methylobacterium extorquens</em> AM1 or <em>Clostridium autoethanogenum</em>, an additional pathway was constructed upon the native pathway to produce L-5-MTHF.</p>
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    <h3>Cultivation, Purification, and SDS-PAGE</h3>
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    <p>The <em>mtdA-fchA</em> 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 <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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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5189/bba-k5189003/1.png" width="40%" alt="Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids">
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        <div style="text-align:center;">
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            <caption>Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids</caption>
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    <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. The SDS-PAGE results displayed distinct bands corresponding to the mtdA-fchA proteins, particularly under induction at 37°C.</p>
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        <img src="https://static.igem.wiki/teams/5189/bba-k5189003/2.png" width="70%" alt="Figure 2: Expression of mtdA-fchA protein in BL21(DE3) analyzed by SDS-PAGE">
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        <div style="text-align:center;">
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            <caption>Figure 2: Expression of mtdA-fchA Protein in BL21(DE3) Analyzed by SDS-PAGE</caption>
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Revision as of 15:29, 27 September 2024


mtdA

mtdA


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 834
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 212
    Illegal NgoMIV site found at 419
  • 1000
    COMPATIBLE WITH RFC[1000]


mtdA Gene Documentation

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.

Figure 1: Gel electrophoresis validation of mtdA-fchA nucleic acids
Figure 1: Gel electrophoresis validation of mtdA-fchA 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. The SDS-PAGE results displayed distinct bands corresponding to the mtdA-fchA proteins, particularly under induction at 37°C.

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