Part:BBa_K5184073

G1M5 tag -PpVP1-F-GNA

Essential Information

Introduction

This composite part is an expression box for the basic part PpVP1F (BBa_K5184038), which allows the PpVP1F-GNA fusion protein, with high contact and oral toxicity, to achieve soluble expression in E. coli. This composite part includes a G1M5-SUMO (BBa_K5184022) compound tag is annexed to the N-terminus of the fusion protein, which facilitates the protein’s correct folding and therefore its soluble expression. GNA, Galanthus nivalis agglutinin (BBa_K1974020) is attached to the C-terminus of the venom peptide, protecting it and conducting it in and through insect guts; achieving high oral and contact toxicity.

Sequences

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]

Characterization

Usage and Biology

G1M5-SUMO-PpVP1F-GNA is a composite part composing of:
G1M5, a extracellular secretion SP utilizing the Sec pathway
SUMO, a protein modifier responsible for assisting folding of the fusion protein, ensures solubility
PpVP1F, a venom peptide from the predatory mite P. persimilis
GNA, a lectin responsible for enhancing oral and contact toxicity of the fusion protein

Toxicity Bioassay

We cooperated with Professor Huang from SCAU for toxicity bioassays. The plasmids are assembled from their respective fragments using GoldenGate cloning. After verification of plasmid constructs via colony PCR and sequencing, we transformed the plasmids into strain BL21(DE3) for expression.
After induction and overnight culture, the culture is harvested and supernatant and precipitate from cell lysis collected. SDS-PAGE is then run to verify soluble expression. The supernatant is subsequentially treated with SUMO protease to remove the SUMO tags.
The bioassay is carried out using a spraying method, where the treated supernatant is sprayed against 3 groups of 20 female T. urticae, and lethality results assessed 24, 48, and 72 hours after initial spraying. The results [Fig1A&B] suggests that the fusion protein achieved significant contact and oral toxicity. Combining with SDS-PAGE results that suggest soluble expression of the protein, all parts of the composite part is functioning as designed.

Fig.1: A. T. urticae before being sprayed with PpVP1F B. T. urticae after being sprayed with PpVP2S C. Survival plot of PpVP1F against female T. urticae using a spraying method, CK is induced liquid culture of BL21(DE3), of which acts as control D. Lethality data of PpVP2S over 24, 48, and 72 hours, CK is induced liquid culture of BL21(DE3), of which acts as control; data is the means of ± SD of three parallel replicate experiments