Part:BBa_K5120015
Hydroxyisoflavanone Dehydratase (HID) in pEAQ DEST 1
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 10226
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 10226
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 5173
Illegal NotI site found at 10232 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 10226
Illegal BglII site found at 644
Illegal BglII site found at 1657
Illegal BglII site found at 1913
Illegal BglII site found at 1933
Illegal BglII site found at 8791
Illegal BglII site found at 9775
Illegal BamHI site found at 1146
Illegal XhoI site found at 714 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 10226
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 10226
Illegal XbaI site found at 10241
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 2397
Illegal NgoMIV site found at 3353
Illegal NgoMIV site found at 3636
Illegal NgoMIV site found at 4168
Illegal NgoMIV site found at 5045
Illegal NgoMIV site found at 5169
Illegal NgoMIV site found at 6196 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Figure 1:Vector Diagram for pmHID in pEAQ DEST 1
pmHID in the pEAQ DEST 1 plasmid is a genetic construct designed for the expression of Hydroxyisoflavanone Dehydratase (HID) in plants like Nicotiana benthamiana. HID plays a crucial role in the biosynthesis of isoflavonoids by catalyzing the dehydration of hydroxyisoflavanones to form isoflavones. This construct is designed to enable efficient expression of pmHID in plant systems, using the pEAQ-HT-DEST1 backbone for high-level transient expression. With its assembly compatibility and regulatory elements, this plasmid serves as an essential tool for studying isoflavonoid biosynthesis and related pathways in synthetic biology.
Features of pEAQ DEST1 plasmid backbone that made us choose it as our plasmid backbone
- Enables transient gene expression in plants
- Contains strong CaMV 35S promoter for high-level expression
- 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
- Suppresses gene silencing via P19 suppressor
- Has kanamycin resistance for transformed bacteria and plant selection
- Contains NOS terminators/promoters commonly used in plant expression
- 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 HID 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 HID 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
- National Center for Biotechnology Information. (n.d.). Nucleotide sequence 257196397. Retrieved from https://www.ncbi.nlm.nih.gov/nuccore/257196397
- 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
- Soo, Sohn In, et al. “Metabolic Engineering of Isoflavones: An Updated Overview.” NCBI, 7 June 2021, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216759/. Accessed 1 October 2024.
| None |