Initially through literature examinations, we have concluded a collection of mite venom peptides from predatory mites that exhibit strong extermination effects on spider mites via targeting calcium channels. All peptides are fused to GNA to achieve contact toxicity, giving BBa_K5184038, BBa_K5184042 , BBa_K5184043. In order to broaden the range of ion channels targetted, we included spider venom peptide in the collection. Thus, we obtained spider venom peptides BBa_K5184021,BBa_K5184032 ,BBa_K5184033 , targeting a variety of ion channels, including voltage-gated sodium channel, calcium ion activated potassium ion channels, and voltage-gated calcium channel. Then we also wanted to enhance the expression success rate of venom peptides, hence incorporating the G1M5-SUMO tag BBa_K5184022. Results show that all venom peptides exhibit strong exterminating effects on female Tetranychidae urticae, a representative of the Tetranychidae mites. We also added new toxicology data to this existing part,BBa_K1974001 , HxTx-Hv1a, a venom peptide from funnel-web spider Hadronyche versus.

Initially through literature examinations, we have concluded a collection of mite venom peptides from predatory mites that exhibit strong extermination effects on spider mites via targeting calcium channels. All peptides are fused to GNA to achieve contact toxicity, giving BBa_K5184038, BBa_K5184042 , BBa_K5184043. In order to broaden the range of ion channels targetted, we included spider venom peptide in the collection. Thus, we obtained spider venom peptides BBa_K5184021,BBa_K5184032 ,BBa_K5184033 , targeting a variety of ion channels, including voltage-gated sodium channel, calcium ion activated potassium ion channels, and voltage-gated calcium channel. Then we also wanted to enhance the expression success rate of venom peptides, hence incorporating the G1M5-SUMO tag BBa_K5184022. Results show that all venom peptides exhibit strong exterminating effects on female Tetranychidae urticae, a representative of the Tetranychidae mites. We also added new toxicology data to this existing part,BBa_K1974001 , HxTx-Hv1a, a venom peptide from funnel-web spider Hadronyche versus.

Design

The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides. [11] [Fig.7A] GNA is fused with the SVPs to enhance oral and contact toxicity. From this, a total of 8 plasmids with mite venom peptide engineered with G1M5, incorporating the SUMO tag were designed, giving G1M5-His-SUMO-MVP-GNA-his. (In which MVP could be NbVP1S, NbVP1F, NbVP2S, NbVP2F, PpVP1S, PpVP1F, PpVP2S, PpVP2F). [Fig.7B]

Build

We employed GoldenGate assembly to assemble our plasmids from respective fragments and the vector pET28a; which were subsequently transformed into the E. coli strain DH5α. The colony PCR results and sequencing results together verifies the plasmid constructs [Fig.8].

Test

For toxicity assay, Professor Huang of SCAU tested the contact toxicity of venom peptides on females of Tetranychus urticae, which is the most prominent species of the spider mites [14]. The SUMO-digested supernatant is applied to the spider mites using a spraying method. The supernatant portion of BL21(DE3) that underwent the same treatments is used as control. The results reveal that PpVP2S, PpVP1S, and PpVP1F are all effective in terms of eliminating T. urticae [Fig.10]. Moreover, PpVP2S achieved a death rate of 98.25% in the first day and 100% in the next.

Learn

Meanwhile, we also learnt that all of our MVPs potentially target the CaV channel. In order to suppress the development of drug resistance, we desire to incorporate SVPs, which target a diverse array of ion channels. Structurally similar, and equally safe to beneficial insects, the SVPs serve as an ideal supplement for MVPs. Since we successfully used the G1M5-SUMO secretion system for MVP biosynthesis, we also plan to use the same expression system for SVPs.

Design

The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides. [11] [Fig.7A] GNA is fused with the SVPs to enhance oral and contact toxicity. From this, a total of 8 plasmids with mite venom peptide engineered with G1M5, incorporating the SUMO tag were designed, giving G1M5-His-SUMO-MVP-GNA-his. (In which MVP could be NbVP1S, NbVP1F, NbVP2S, NbVP2F, PpVP1S, PpVP1F, PpVP2S, PpVP2F). [Fig.7B]

Build

We employed GoldenGate assembly to assemble our plasmids from respective fragments and the vector pET28a; which were subsequently transformed into the E. coli strain DH5α. The colony PCR results and sequencing results together verifies the plasmid constructs [Fig.8].

Test

For toxicity assay, Professor Huang of SCAU tested the contact toxicity of venom peptides on females of Tetranychus urticae, which is the most prominent species of the spider mites [14]. The SUMO-digested supernatant is applied to the spider mites using a spraying method. The supernatant portion of BL21(DE3) that underwent the same treatments is used as control. The results reveal that PpVP2S, PpVP1S, and PpVP1F are all effective in terms of eliminating T. urticae [Fig.10]. Moreover, PpVP2S achieved a death rate of 98.25% in the first day and 100% in the next.

Learn

Meanwhile, we also learnt that all of our MVPs potentially target the CaV channel. In order to suppress the development of drug resistance, we desire to incorporate SVPs, which target a diverse array of ion channels. Structurally similar, and equally safe to beneficial insects, the SVPs serve as an ideal supplement for MVPs. Since we successfully used the G1M5-SUMO secretion system for MVP biosynthesis, we also plan to use the same expression system for SVPs.

Design

The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides. [11] [Fig.7A] GNA is fused with the SVPs to enhance oral and contact toxicity. From this, a total of 8 plasmids with mite venom peptide engineered with G1M5, incorporating the SUMO tag were designed, giving G1M5-His-SUMO-MVP-GNA-his. (In which MVP could be NbVP1S, NbVP1F, NbVP2S, NbVP2F, PpVP1S, PpVP1F, PpVP2S, PpVP2F). [Fig.7B]

Build

We employed GoldenGate assembly to assemble our plasmids from respective fragments and the vector pET28a; which were subsequently transformed into the E. coli strain DH5α. The colony PCR results and sequencing results together verifies the plasmid constructs [Fig.8].

Test

For toxicity assay, Professor Huang of SCAU tested the contact toxicity of venom peptides on females of Tetranychus urticae, which is the most prominent species of the spider mites [14]. The SUMO-digested supernatant is applied to the spider mites using a spraying method. The supernatant portion of BL21(DE3) that underwent the same treatments is used as control. The results reveal that PpVP2S, PpVP1S, and PpVP1F are all effective in terms of eliminating T. urticae [Fig.10]. Moreover, PpVP2S achieved a death rate of 98.25% in the first day and 100% in the next.

Learn

Meanwhile, we also learnt that all of our MVPs potentially target the CaV channel. In order to suppress the development of drug resistance, we desire to incorporate SVPs, which target a diverse array of ion channels. Structurally similar, and equally safe to beneficial insects, the SVPs serve as an ideal supplement for MVPs. Since we successfully used the G1M5-SUMO secretion system for MVP biosynthesis, we also plan to use the same expression system for SVPs.