Difference between revisions of "Part:BBa K5120004"

 
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<partinfo>BBa_K5120004 short </partinfo>
  
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===Sequence and Features===
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<partinfo>BBa_K5120004 SequenceAndFeatures</partinfo>
  
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<h2>Usage and Biology</h2>
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<h6>Figure 1: Isoflavonoid Biosynthetic Pathway</h6>
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C-glycosyltransferase (CGT) in the pEAQ DEST 1 plasmid is a genetic construct designed for the expression of the CGT enzyme from Pueraria Mirifica Var. Lobata in plants like Nicotiana benthamiana. CGT catalyzes the conversion of flavanones into isoflavones, which are key compounds in the biosynthesis of isoflavonoids. This construct, which is 1566 base pairs long, is optimized for efficient expression in plant systems, using the pEAQ-HT-DEST1 backbone for high-level transient expression. With its assembly compatibility and regulatory elements, it serves as a powerful tool for exploring isoflavonoid biosynthesis and metabolic engineering in synthetic biology.
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The enzyme C-glycosyltransferase (CGT)  from Pueraria Mirifica Var. Lobata is involved in the biosynthesis of isoflavonoids. CGT catalyzes the dehydration of 2-hydroxyisoflavonones, facilitating the formation of isoflavonoid compounds. This enzyme part is designed to be compatible with assembly standards for incorporation into various systems for further research or synthetic biology applications. It is 972 base pairs long and is compatible with several assembly standards, allowing it to be integrated into constructs for expression in plants like Nicotiana benthamiana.
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<h2>Proof of Function</h2>
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<p>This part was used in a composite part along with other key enzymes in the isoflavonoid biosynthetic pathway and was agroinfiltrated into <i>N. benthamiana</i> using Agrobacterium tumefaciens.
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The enzyme C-glycosyltransferase (CGT) from Pueraria Mirifica Var. Lobata is involved in the glycosylation of flavonoids by attaching sugar molecules to the flavonoid backbone through C-glycosylation. This process is important in the biosynthesis of glycosylated flavonoids, which have enhanced stability and solubility in plants. The CGT part is 1410 base pairs long and is designed to be compatible with several assembly standards, making it suitable for the use in synthetic biology projects to express this enzyme in hose organisms such as NIcotiana benthamiana for further research on flavonoid biosynthesis.
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<img class="figure" src="https://static.igem.wiki/teams/5120/part-registry/pcr-results.png">
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<h6>Figure 2: PCR results for composite parts with genes for selected enzymes from the Isoflavonoid biosynthetic pathway</h6>
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<img class="figure" src="https://static.igem.wiki/teams/5120/part-registry/hplc-analysis.png">
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<h6>Figure 3: HPLC Chromatogram showing the detection of puerarin, daidzin, genistin, iso-vitexin, daidzien and genistein in transformed <i>Nicotiana benthamiana</i> samples</h6>
  
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After transformation, the modified plants were tested for isoflavonoid production using High-Performance Liquid Chromatography (HPLC). The chromatogram shows the amounts of each target isoflavonoid: puerarin, daidzein, and genistein with the first peak, observed at around 16.0 minutes, representing puerarin, followed by a peak at approximately 17.0 minutes, which corresponds to daidzin. Further along, a peak at 22.0 minutes is attributed to genistin. Traces of all three compounds were detected in <i>N. benthamiana</i>, a plant that does not naturally produce any of these because it lacks the enzymes needed to do so. This shows that the sequence for CGT did function as intended because if it hadn't then the pathway wouldn't have progressed further and produced these isoflavonoids.
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===Functional Parameters===
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Revision as of 19:55, 1 October 2024

C-glycosyltransferase (CGT) in Pueraria Mirifica Var. Lobata

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Figure 1: Isoflavonoid Biosynthetic Pathway

C-glycosyltransferase (CGT) in the pEAQ DEST 1 plasmid is a genetic construct designed for the expression of the CGT enzyme from Pueraria Mirifica Var. Lobata in plants like Nicotiana benthamiana. CGT catalyzes the conversion of flavanones into isoflavones, which are key compounds in the biosynthesis of isoflavonoids. This construct, which is 1566 base pairs long, is optimized for efficient expression in plant systems, using the pEAQ-HT-DEST1 backbone for high-level transient expression. With its assembly compatibility and regulatory elements, it serves as a powerful tool for exploring isoflavonoid biosynthesis and metabolic engineering in synthetic biology. .

The enzyme C-glycosyltransferase (CGT) from Pueraria Mirifica Var. Lobata is involved in the biosynthesis of isoflavonoids. CGT catalyzes the dehydration of 2-hydroxyisoflavonones, facilitating the formation of isoflavonoid compounds. This enzyme part is designed to be compatible with assembly standards for incorporation into various systems for further research or synthetic biology applications. It is 972 base pairs long and is compatible with several assembly standards, allowing it to be integrated into constructs for expression in plants like Nicotiana benthamiana.

Proof of Function

This part was used in a composite part along with other key enzymes in the isoflavonoid biosynthetic pathway and was agroinfiltrated into N. benthamiana using Agrobacterium tumefaciens.

The enzyme C-glycosyltransferase (CGT) from Pueraria Mirifica Var. Lobata is involved in the glycosylation of flavonoids by attaching sugar molecules to the flavonoid backbone through C-glycosylation. This process is important in the biosynthesis of glycosylated flavonoids, which have enhanced stability and solubility in plants. The CGT part is 1410 base pairs long and is designed to be compatible with several assembly standards, making it suitable for the use in synthetic biology projects to express this enzyme in hose organisms such as NIcotiana benthamiana for further research on flavonoid biosynthesis.

Figure 2: PCR results for composite parts with genes for selected enzymes from the Isoflavonoid biosynthetic pathway

Figure 3: HPLC Chromatogram showing the detection of puerarin, daidzin, genistin, iso-vitexin, daidzien and genistein in transformed Nicotiana benthamiana samples

After transformation, the modified plants were tested for isoflavonoid production using High-Performance Liquid Chromatography (HPLC). The chromatogram shows the amounts of each target isoflavonoid: puerarin, daidzein, and genistein with the first peak, observed at around 16.0 minutes, representing puerarin, followed by a peak at approximately 17.0 minutes, which corresponds to daidzin. Further along, a peak at 22.0 minutes is attributed to genistin. Traces of all three compounds were detected in N. benthamiana, a plant that does not naturally produce any of these because it lacks the enzymes needed to do so. This shows that the sequence for CGT did function as intended because if it hadn't then the pathway wouldn't have progressed further and produced these isoflavonoids.