Difference between revisions of "Part:BBa K5184071"
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<partinfo>BBa_K5184071 short</partinfo> | <partinfo>BBa_K5184071 short</partinfo> | ||
| − | + | ==Essential Information== | |
| + | ===Introduction=== | ||
| + | This composite part is an expression box for the basic part PpVP2S (BBa_K5184043), which allows the PpVP2S-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=== | |
| − | === | + | |
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<partinfo>BBa_K5184071 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5184071 SequenceAndFeatures</partinfo> | ||
| + | ==Characterization== | ||
| + | ===Usage and Biology=== | ||
| + | G1M5-SUMO-PpVP2S-GNA is a composite part composing of:<br> | ||
| + | G1M5, a extracellular secretion SP utilizing the Sec pathway<br> | ||
| + | SUMO, a protein modifier responsible for assisting folding of the fusion protein, ensures solubility<br> | ||
| + | PpVP2S, a venom peptide from the predatory mite ''P. persimilis''<br> | ||
| + | GNA, a lectin responsible for enhancing oral and contact toxicity of the fusion protein<br> | ||
| + | |||
| + | ===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.<br> | ||
| + | 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.<br> | ||
| + | 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. | ||
| + | <center><html><img src="https://static.igem.wiki/teams/5184/parts/ppvp2s-lethality.webp" width="600"/></html></center> | ||
| + | <center><b>Fig.1: A. ''T. urticae'' before being sprayed with PpVP2S B. ''T. urticae'' after being sprayed with PpVP2S C. Survival plot of PpVP2S 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</b></center> | ||
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Latest revision as of 11:27, 2 October 2024
G1M5 tag-PpVP2-S-GNA
Essential Information
Introduction
This composite part is an expression box for the basic part PpVP2S (BBa_K5184043), which allows the PpVP2S-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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
Usage and Biology
G1M5-SUMO-PpVP2S-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
PpVP2S, 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.
