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Chalcone Synthase in pEAQ HT DES1

Sequence and Features

Usage and Biology

Figure 1:Vector Diagram for pmCHS in pEAQ DEST 1

pmCHS in the pEAQ DEST 1 plasmid is a genetic construct designed for the expression of the Chalcone Synthase (CHS) enzyme in plants like Nicotiana benthamiana. CHS catalyzes the first step in the biosynthesis of flavonoids by converting p-coumaroyl-CoA and malonyl-CoA into naringenin chalcone, a precursor for various flavonoid compounds. This construct is tailored to ensure efficient expression of pmCHS in plant systems, using the pEAQ-HT-DEST1 backbone for high-level transient expression. With its assembly compatibility and regulatory elements, it is a useful tool for studying flavonoid production and metabolic pathways in synthetic biology.

Features of pEAQ DEST1 plasmid backbone that made us choose it as our plasmid backbone

1. Enables transient gene expression in plants
2. Contains strong CaMV 35S promoter for high-level expression
3. Is equipped with elements such as the LB and RB T-DNA repeats, which are necessary for T-DNA transfer during Agrobacterium-mediated plant transformation
4. Suppresses gene silencing via P19 suppressor
5. Has kanamycin resistance for transformed bacteria and plant selection
6. Contains NOS terminators/promoters commonly used in plant expression
7. Is Compatible with gateway in-fusion cloning technology

Proof of Function

To allow for transient (co)expression of isoflavone biosynthetic genes in N. benthamiana, we utilized agrobacterium vector-mediated infiltration (commonly referred to as agro-infiltration) as a method to perform transient transformation.

Figure 2: Primers used for colony PCR

This composite part was first cloned using In-Fusion Cloning where the target gene fused with the plasmid backbone. From then on, the resulting solution (from the combination phase of In-Fusion Cloning) was transformed into Escherichia coli strain DH5-α via the heat shock transformation method. Then the transformed E. coli strain was grown in Kanamycin to verify transformation and transformed E. coli strains were picked through colony PCR. The transformed plasmids in the E. coli strains were introduced to A. tumefaciens where the A. tumefaciens was again grown.

Figure 3: PCR results for composite parts with genes for selected enzymes from the Isoflavonoid biosynthetic pathway

The Polymerase Chain Reaction (PCR) was used to confirm the successful integration of the composite part into the N. benthamiana genome after agroinfiltration. 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 in the composite part, 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 corresponding 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 composite part did function as intended because if it hadn't then the pathway wouldn't have progressed further and produced these isoflavonoids.

References

1. National Center for Biotechnology Information. (n.d.). Nucleotide sequence 257196397. Retrieved from https://www.ncbi.nlm.nih.gov/nuccore/257196397
2. iGEM Parts Registry. (n.d.). Help: Plasmid backbones/Entering new plasmids in the Registry. Retrieved from https://parts.igem.org/Help:Plasmid_backbones/Entering_new_plasmids_in_the_Registry
3. 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/.