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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In order to eliminate T.urticae of infested cultivations, spider venom peptide rCtx-4 is incorporated in our project with pesticidal means. rCtx-4 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. Due to the cysteine-rich nature of rCtx-4, expression strategy that exterminates inclusion bodies is required, thus a G1M5 secretion system is engineered with the SVP to achieve this. Considering future pesticidal control, G1M5-rCtx-4-his can provide future iGEM teams that dedicate in exterminating other destructive pests more choices of sustainable pesticide that could be expressed correctly in Escherichia Coli.  
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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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===Usage and Biology===
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===Sequences===
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rCtx-4 is a small cysteine-rich venom peptide derived from Phoneutria depilate, consisting of 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. The sequence of it is annotated in the transcriptome as a sodium channel neurotoxin. In nature, utilized as predatory toxin, it carries the ability to cause paralysis and subsequent death in susceptible subjects by acting on the voltage-gated sodium channel (Nav) of susceptible subjects. G1M5 is the mutated, less hydrophobic version of the secretion signal peptide of the G1 cyclomaltodextrin glucanotransferase (CGtase) of Bacillus sp., which allows the extracellular secretion of the bacterial enzyme. Conduction of proteins attached by G1M5 out of the cytosol is achieved by the Sec pathway, a very common secretion system seen in all three major domains of life: arachaea, prokaryote, and eukaryotes. Once the signal peptide, in this case G1M5 is synthesized, the protein chaperon SecB binds to the preprotein (that is attached to G1M5), and transfers the preprotein to the protein translocase SecA, of which binds to the membrane bound protein conducting channel SecYEG. Once bound to the membrane, SecA binds to a molecule of ATP, of which is hydrolyzed to conduct the protein through heterotrimer complex of SecYEG. A membrane bound SPaseI then, once enough of the preprotein had been conducted through the channel, will remove the SP and allow the preprotein to fold properly into the correct protein.
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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