Difference between revisions of "Part:BBa K5120002"

Revision as of 09:09, 2 October 2024

Isoflavone Synthase (IFS) in Pueraria Mirifica Var. Lobata

Sequence and Features

Usage and Biology

Figure 1: Isoflavonoid Biosynthetic Pathway

Isoflavone Synthase (IFS) in the pEAQ DEST 1 plasmid is a genetic construct designed for the expression of the IFS enzyme from Pueraria Mirifica Var. Lobata in plants like Nicotiana benthamiana. IFS 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. .

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 Isoflavone Synthase(IFS) 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 IFS 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 IFS 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 IFS did function as intended because if it hadn't then the pathway wouldn't have progressed further and produced these isoflavonoids.