Difference between revisions of "Part:BBa K5184021"

 
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<partinfo>BBa_K5184021 short</partinfo>
 
<partinfo>BBa_K5184021 short</partinfo>
  
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"Ctx-4 is an insecticidal peptide obtained from the ctenid spider Phoneutria depilata. It is consists of 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. It's a venom peptide used by P. depilata to incapacitate their prey. Based on its original amino acid sequence, a synthetic gene is assembled through the use of overlapping PCR. The new gene is thus called recombinant Ctx-4, also abbreviated as rCtx-4. The sequence of it is annotated in the transcriptome as a sodium channel neurotoxin. However, during in vivo experiments, rCtx-4 is proven to only establish paralyzing effects on certain invertabrates such as crickets and mites. Meanwhile, mammals including mice were not affected by its toxicity.
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In order to eliminate spider mites, the spider venom peptide rCtx-4 is incorporated in our project to broaden the spectrum of molecular targets of other venom peptides. rCtx4 is a neurotoxin obtained from the ctenid spider ''Phoneutria depilata'', naturally employed to incapacitate their prey. Having the voltage-gated sodium channels playing a vital role in neuronal, muscular and cardiac functions, targets experience fast immobilization after envenomation, thus it serves as an effective pesticide.
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In our context, we attempted to construct the rCtx-4 sequence onto the vector pQE30 and transform it into E.coli to express the venom peptide. The venom peptide obtained is purified and injected into Tetrachynus urticae to establish paralyzing effects."
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===Usage and Biology===
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===Sequences===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5184021 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5184021 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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rCtx-4 is a small cysteine-rich venom peptide derived from Phoneutria depilate, first identified in [1], it consists of 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. It has a cysteine network of C1xxxC2xxxC3xC4C5xxxC6xC7xxxC8xC9xxxC10, with disulfide bridges between C1C5, C2C6, C3C10, C4C9, and C7C8 [Fig1 A&B]. In addition to the covalent disulfide bridges, there is also a pair of beta strands that run antiparallel to each other, allowing hydrogen bonds between each other and, together with the disulfide bonds, folds the protein into a highly compact conformation.
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/rctx4-structure.webp" width="600"/></html></center>
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<center><b>Fig1: (A) Cysteine cross-bridge structure in rCtx4 B. Secondary structure of rCtx4, by structural prediction results from AlphaFold. The cyestine residues are colored orange, displaying their side chains and the rest of the peptide in white</b></center>
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Such a compact conformation gives rise to an inhibitor-cysteine-knot (ICK) structure between C6 and C5, allowing the venom peptide to have inhibitory effects on its molecular target: voltage-gated sodium ion channels, and to have a highly stable structure.
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===Toxicity Verification===
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rCtx4 is synthesized using the vector pET28a-G1M5-His-SUMO-rCtx4-GNA-His[Fig2B], of which is assembled using GoldenGate cloning and transformed into ''E. coli'' strain DH5ɑ. Colony PCR and sequencing is then carried out to verify the plasmid construct, of which is extracted and transformed into BL21(DE3) strain for expression. After IPTG induction and overnight incubation, the liquid culture is harvested and, after cell lysis, have an SDS-PAGE run. The results suggest that rCtx4 had achieved soluble expression. After several unsuccessful purification attempts, the supernatant is treated directly by SUMO protease [Fig2C].
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/rctx4-sumo.webp" width="600"/></html></center>
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<center><b>Fig2: (A) G1M5 tag allows secretion of the fusion protein into extracellular milieu (B) Plasmid construct pET28a-G1M5-His-SUMO-rCtx4-GNA-His (C) SDS-PAGE of supernatant and SUMO-treated supernatant, with supernatant of similarly treated supernatant of BL21(DE3) as control</b></center>
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The SUMO-digested supernatant's toxicity against ''T. urticae'' females is tested using a spraying method by Professor Huang from SCAU. Results from the toxicity assay suggests rCtx4 to be highly toxic against ''T. urticae'', achieving an astonishing fatality of 96.49% within 24 hours and complete elimination after another day [Fig2C&D].
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/rctx4-lethality1.webp" width="600"/></html></center>
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<center><b>Fig3: (A) ''T. urticae'' in their normal state, before being sprayed with treated supernatant (B) Dead ''T. urticae'' from spraying of treated supernatant (C) Survival plot of ''T. urticae'' being sprayed with supernatant containing rCtx4 over 72 hours, CK is similarly processed supernatant of BL21(DE3), acting as a control (D) Lethality data of ''T. urticae'' being sprayed with supernatant over 24, 48, and 72 hours, CK is the similarly processed supernatant of BL21(DE3), acting as a control</b></center>
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==Part Collection==
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Our part collection provides a comprehensive list of venom peptides with a diverse range of molecular targets, and all displays satisfactory elimination efficacy during our testings [Fig7A&B].
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/vp-lethality-v.webp" width="600"/></html></center>
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<center><b>Fig4: A. Survival plot of 6 venom peptides 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 6 venom peptides 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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{|class="wikitable" style="margin:auto"
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|+ Our Part Collection
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|-
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!Current VP!!Venom Name!!Targeted Ion Channel!!New?!!Part Number!!Original Specie
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|-
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|||PpVP2S||Ca||New||BBa_K5184043||''Phytoseiulus persimilis''
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|-
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|||PpVP1S||Ca||New||BBa_K5184042||''Phytoseiulus persimilis''
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|-
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|||PpVP1F||Ca||New||BBa_K5184038||''Phytoseiulus persimilis''
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|-
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|✳️||rCtx4||Na||||BBa_K5184021||''Phoneutria depilata''
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|-
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|||Cs1A||Ca||||BBa_K5184032||''Calommata signata''
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|-
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|||HxTx-Hv1h||Ca, K||||BBa_K5184033||''Hadronyche versuta''
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|}
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===References===
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[1]: Vásquez-Escobar, J.;  Benjumea-Gutiérrez, D.M.; Lopera,  C.; Clement, H.C.; Bolaños, D.I.;  Higuita-Castro, J.L.; Corzo, G.A.;  Corrales-Garcia, L.L. Heterologous  Expression of an Insecticidal Peptide  Obtained from the Transcriptome of  the Colombian Spider Phoneutria  depilate. Toxins 2023, 15, 436.  https://doi.org/10.3390/toxins15070436
  
 
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Latest revision as of 12:17, 2 October 2024


rCtx-4

In order to eliminate spider mites, the spider venom peptide rCtx-4 is incorporated in our project to broaden the spectrum of molecular targets of other venom peptides. rCtx4 is a neurotoxin obtained from the ctenid spider Phoneutria depilata, naturally employed to incapacitate their prey. Having the voltage-gated sodium channels playing a vital role in neuronal, muscular and cardiac functions, targets experience fast immobilization after envenomation, thus it serves as an effective pesticide.

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]

Usage and Biology

rCtx-4 is a small cysteine-rich venom peptide derived from Phoneutria depilate, first identified in [1], it consists of 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. It has a cysteine network of C1xxxC2xxxC3xC4C5xxxC6xC7xxxC8xC9xxxC10, with disulfide bridges between C1C5, C2C6, C3C10, C4C9, and C7C8 [Fig1 A&B]. In addition to the covalent disulfide bridges, there is also a pair of beta strands that run antiparallel to each other, allowing hydrogen bonds between each other and, together with the disulfide bonds, folds the protein into a highly compact conformation.

Fig1: (A) Cysteine cross-bridge structure in rCtx4 B. Secondary structure of rCtx4, by structural prediction results from AlphaFold. The cyestine residues are colored orange, displaying their side chains and the rest of the peptide in white

Such a compact conformation gives rise to an inhibitor-cysteine-knot (ICK) structure between C6 and C5, allowing the venom peptide to have inhibitory effects on its molecular target: voltage-gated sodium ion channels, and to have a highly stable structure.

Toxicity Verification

rCtx4 is synthesized using the vector pET28a-G1M5-His-SUMO-rCtx4-GNA-His[Fig2B], of which is assembled using GoldenGate cloning and transformed into E. coli strain DH5ɑ. Colony PCR and sequencing is then carried out to verify the plasmid construct, of which is extracted and transformed into BL21(DE3) strain for expression. After IPTG induction and overnight incubation, the liquid culture is harvested and, after cell lysis, have an SDS-PAGE run. The results suggest that rCtx4 had achieved soluble expression. After several unsuccessful purification attempts, the supernatant is treated directly by SUMO protease [Fig2C].

Fig2: (A) G1M5 tag allows secretion of the fusion protein into extracellular milieu (B) Plasmid construct pET28a-G1M5-His-SUMO-rCtx4-GNA-His (C) SDS-PAGE of supernatant and SUMO-treated supernatant, with supernatant of similarly treated supernatant of BL21(DE3) as control

The SUMO-digested supernatant's toxicity against T. urticae females is tested using a spraying method by Professor Huang from SCAU. Results from the toxicity assay suggests rCtx4 to be highly toxic against T. urticae, achieving an astonishing fatality of 96.49% within 24 hours and complete elimination after another day [Fig2C&D].

Fig3: (A) T. urticae in their normal state, before being sprayed with treated supernatant (B) Dead T. urticae from spraying of treated supernatant (C) Survival plot of T. urticae being sprayed with supernatant containing rCtx4 over 72 hours, CK is similarly processed supernatant of BL21(DE3), acting as a control (D) Lethality data of T. urticae being sprayed with supernatant over 24, 48, and 72 hours, CK is the similarly processed supernatant of BL21(DE3), acting as a control

Part Collection

Our part collection provides a comprehensive list of venom peptides with a diverse range of molecular targets, and all displays satisfactory elimination efficacy during our testings [Fig7A&B].

Fig4: A. Survival plot of 6 venom peptides 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 6 venom peptides 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
Our Part Collection
Current VP Venom Name Targeted Ion Channel New? Part Number Original Specie
PpVP2S Ca New BBa_K5184043 Phytoseiulus persimilis
PpVP1S Ca New BBa_K5184042 Phytoseiulus persimilis
PpVP1F Ca New BBa_K5184038 Phytoseiulus persimilis
✳️ rCtx4 Na BBa_K5184021 Phoneutria depilata
Cs1A Ca BBa_K5184032 Calommata signata
HxTx-Hv1h Ca, K BBa_K5184033 Hadronyche versuta


References

[1]: Vásquez-Escobar, J.; Benjumea-Gutiérrez, D.M.; Lopera, C.; Clement, H.C.; Bolaños, D.I.; Higuita-Castro, J.L.; Corzo, G.A.; Corrales-Garcia, L.L. Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate. Toxins 2023, 15, 436. https://doi.org/10.3390/toxins15070436