Difference between revisions of "Part:BBa K5120000"
 
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<partinfo>BBa_K5120000 short </partinfo>
  
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__NOTOC__
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===Sequence and Features===
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<partinfo>BBa_K5120000 short</partinfo>
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<partinfo>BBa_K5120000 SequenceAndFeatures</partinfo>
  
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<!-- Add more about the biology of this part here -->
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<h2>Usage and Biology</h2>
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===Usage and Biology===
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[[Image:isoflavanoid-pathway-fused.jpg|450px|thumb|right|'''Figure 1:''' Isoflavonoid Biosynthetic Pathway, scale: 3&nbsp;L, [https://tools.igem.org/uploads/teams/5120/generalimgs]]]
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<img class="figure" src="https://static.igem.wiki/teams/5120/projectdescription1.png">
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<h6>Figure 1: Isoflavonoid Biosynthetic Pathway</h6>
  
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Chalcone Synthase(CHS) is an enzyme that catalyzes the first step in the production of flavonoids, converting p-coumaroyl-CoA and malonyl-CoA into naringenin chalcone or isoliquiritigenin, which is the precursor for various flavonoid compounds, including flavanones, flavonols, and anthocyanins.
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<p>
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Chalcone Synthase (CHS) enzyme, derived from <i>Pueraria Mirifica Var. Lobata</i>, catalyzes the first step in the biosynthesis of flavonoids. It converts p-coumaroyl-CoA and malony-COA into naringenin chalcone or isoliquiritigenin, which are precursors to various flavonoids compounds like flavnones, flavonols and anthocyanins. This part is engineered for use in <i>Nicotiana benthamiana</i> to express CHS for the production of isoflavonoids. After transformation using <i>Agrobacterium tumefaciens</i>, successful production of isoflavonoids such as puerarin, daidzein and genistein was confirmed through HPLC analysis.
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It is constructed for use in plants, specifically <i>Nicotiana benthamiana</i>, to be included in a composite part that allows for the expression of CHS in the biosynthesis of isoflavonoids.
 
  
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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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<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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First, Polymerase Chain Reaction (PCR) was used to confirm the successful integration of the Chalcone Synthase (CHS) gene into the <i>Nicotiana benthamiana</i> genome after agroinfiltration.(Results seen in Figure 2) PCR amplifies specific DNA sequences, allowing researchers to detect where the gene of interest can be inserted into the plant's genome. In this case, primers were designed to target the CHS gene, and after running the PCR, the amplified product was visualized on an agarose gel. A distinct band corresponding to the expected size of the CHS gene confirmed that the transformation was successful, further supporting the functionality of the pathway and the production of isoflavonoids in the modified plants.
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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 CHS 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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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5120000 SequenceAndFeatures</partinfo>
 
  
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<h2>References</h2>
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<li>Dao, T T H, et al. “Chalcone Synthase and Its Functions in Plant Resistance.” Phytochemistry Reviews : Proceedings of the Phytochemical Society of Europe, U.S. National Library of Medicine, Sept. 2011, www.ncbi.nlm.nih.gov/pmc/articles/PMC3148432/. </li>
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===Functional Parameters===
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<partinfo>BBa_K5120000 parameters</partinfo>
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Latest revision as of 09:46, 2 October 2024

Chalcone Synthase (CHS) 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

Chalcone Synthase (CHS) enzyme, derived from Pueraria Mirifica Var. Lobata, catalyzes the first step in the biosynthesis of flavonoids. It converts p-coumaroyl-CoA and malony-COA into naringenin chalcone or isoliquiritigenin, which are precursors to various flavonoids compounds like flavnones, flavonols and anthocyanins. This part is engineered for use in Nicotiana benthamiana to express CHS for the production of isoflavonoids. After transformation using Agrobacterium tumefaciens, successful production of isoflavonoids such as puerarin, daidzein and genistein was confirmed through HPLC analysis. .

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.

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

First, Polymerase Chain Reaction (PCR) was used to confirm the successful integration of the Chalcone Synthase (CHS) gene into the Nicotiana benthamiana genome after agroinfiltration.(Results seen in Figure 2) PCR amplifies specific DNA sequences, allowing researchers to detect where the gene of interest can be inserted into the plant's genome. In this case, primers were designed to target the CHS gene, and after running the PCR, the amplified product was visualized on an agarose gel. A distinct band corresponding to the expected size of the CHS gene confirmed that the transformation was successful, further supporting the functionality of the pathway and the production of isoflavonoids in the modified plants.

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 CHS did function as intended because if it hadn't then the pathway wouldn't have progressed further and produced these isoflavonoids.

References

  1. Dao, T T H, et al. “Chalcone Synthase and Its Functions in Plant Resistance.” Phytochemistry Reviews : Proceedings of the Phytochemical Society of Europe, U.S. National Library of Medicine, Sept. 2011, www.ncbi.nlm.nih.gov/pmc/articles/PMC3148432/.