Plasmid_Backbone

Part:BBa_K5120006

Designed by: Ananya Anuj Dharna   Group: iGEM24_Thailand-RIS   (2024-10-01)
Revision as of 09:26, 2 October 2024 by Ananyad (Talk | contribs)

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pEAQ-HT-DEST1 Plasmid Backbone

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 10226
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 10226
    Plasmid lacks a suffix.
    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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.

Usage and Biology

Figure 1: pEAQ DEST 1 vector diagram

The pEAQ-HT-DEST1 plasmid backbone is a cloning vector designed for high-level transient expression of proteins in plants. It includes important features like the CaMV 35S promoter for strong expression, a P19 silencer to suppress post-transcriptional gene silencing, and a Neo/KanR resistance gene for selection in bacterial systems. The plasmid is 10,247 base pairs long and is used as a backbone for constructing vectors that can express desired genes in plants, such as Nicotiana benthamiana, for experiments in synthetic biology. It is compatible with several assembly standards, allowing for flexible cloning and expression applications.

Features of pEAQ DEST1 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
    8. li>Has a Biobrick prefix and suffix at its ends

Proof of Function

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

Figure 2: 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(Figure 2) 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 plasmid did function as intended because if it hadn't then the pathway wouldn't have been recreated 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

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