Difference between revisions of "Part:BBa K5184033"
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<partinfo>BBa_K5184033 short</partinfo>
 
<partinfo>BBa_K5184033 short</partinfo>
  
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In order to eliminate T.urticae of infested cultivations, spider venom peptide G1M5-HxTx-Hv1h-his is incorporated in our project with pesticidal means. HxTx-Hv1h is a small cysteine-rich venom peptide derived from Blue mountains funnel-web spiders Hadronyche versuta. In nature, utilized as predatory toxin, it carries the ability to cause paralysis and death in susceptible subjects by targeting both calcium ion channels and potassium ion channels of them. Though its strong activity on certain invertebrates, it is proven no harm to non-pest insects such as Dalotia coriaria, Orius insidiosus, and pollinating bees of genus Apis. Having both ion channels playing a fundamental role in the neuronal system, targets experience fast immobilization after envenomation, thus it serves as an effective pesticide. Due to the cysteine-rich nature of HxTx-Hv1h, expression strategy that exterminates inclusion bodies is required, thus a G1M5 secretion system is engineered with HxTx-Hv1h to achieve this. Considering future pesticidal control, G1M5-HxTx-Hv1h-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, spider venom peptide HxTx-Hv1h is incorporated in our project to broaden the spectrum of molecular targets of venom peptides. HxTx-Hv1h is a small cysteine-rich venom peptide derived from <i>Hadronyche versuta</i>. Utilized as predatory toxin in nature, it carries the ability to cause paralysis and death in susceptible subjects by interfering with voltage-gated calcium and potassium channels. Given CaV and KV channels' roles in neurotransmitter release and neural impulse relay, HxTx-Hv1h serves as an effective pesticide.
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===Usage and Biology===
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Paralysis and mortal effects of susceptible subjects are achieved via HxTx-Hv1h target on nicotinic Acetylcholine receptors (nAChRs), voltage-activated calcium ion channel (Cav) and calcium ion activated potassium ion channels (Kca) of targets. 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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===Sequences===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5184033 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5184033 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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HxTx-Hv1h is a 39aa long peptide containing 6 cysteine residues arranged to create a cysteine framework of C1xxxC2xxxC3C4xxxC5xxxC6, forming cysteine cross bridges between C1C4, C2C6, and between C3C5 [Fig1A].
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/hv1h-structure.webp" width="600"/></html></center>
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<center><b>Fig1: (A) Cysteine cross-bridge structure in HxTx-Hv1h (B) Secondary structure of HxTx-Hv1h, by structural prediction results from AlphaFold. The cysteine residues are colored orange, displaying their side chains and the rest of the peptide in white</b></center>
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Paralysis and mortal effects of susceptible subjects are achieved via HxTx-Hv1h inhibition on both CaV and KV channels of susceptible targets by blocking the channels directly, resulting in impediment of fundamental nervous system responses in neuronal, muscular, and cardiac functions. (Specific site of inhibition depends on the concentration of neurotoxin).
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===Toxicity Verification===
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HxTx-Hv1h is synthesized using the vector pTE28a-G1M5-His-SUMO-HxTx-Hv1h-GNA-His[Fig2B], of which is assembled using GoldenGate cloning and transformed into <i>E. coli</i> 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 HxTx-Hv1h 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/hv1h-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-HxTx-Hv1h-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 <i>T. urticae</i> females is tested using a spraying method by Professor Huang from SCAU.
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Results from the toxicity assay suggests HxTx-Hv1h to be highly toxic against <i>T. urticae</i>, achieving an fatality of 90.91% within 72 hours.
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<center><html><img src="https://static.igem.wiki/teams/5184/parts/hv1h-lethality.webp" width="600"/></html></center>
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<center><b>Fig2: (A) <i>T. urticae</i> in their normal state, before being sprayed with treated supernatant (B) Dead <i>T. urticae</i> from spraying of treated supernatant (C) Survival plot of <i>T. urticae</i> being sprayed with supernatant containing HxTx-Hv1h over 72 hours, CK is similarly processed supernatant of BL21(DE3), acting as a control (D) Lethality data of <i>T. urticae</i> 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.webp" width="600"/></html></center>
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<center><b>Fig7: 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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Revision as of 08:08, 2 October 2024


HxTx-Hv1h

In order to eliminate spider mites, spider venom peptide HxTx-Hv1h is incorporated in our project to broaden the spectrum of molecular targets of venom peptides. HxTx-Hv1h is a small cysteine-rich venom peptide derived from Hadronyche versuta. Utilized as predatory toxin in nature, it carries the ability to cause paralysis and death in susceptible subjects by interfering with voltage-gated calcium and potassium channels. Given CaV and KV channels' roles in neurotransmitter release and neural impulse relay, HxTx-Hv1h 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

HxTx-Hv1h is a 39aa long peptide containing 6 cysteine residues arranged to create a cysteine framework of C1xxxC2xxxC3C4xxxC5xxxC6, forming cysteine cross bridges between C1C4, C2C6, and between C3C5 [Fig1A].

Fig1: (A) Cysteine cross-bridge structure in HxTx-Hv1h (B) Secondary structure of HxTx-Hv1h, by structural prediction results from AlphaFold. The cysteine residues are colored orange, displaying their side chains and the rest of the peptide in white

Paralysis and mortal effects of susceptible subjects are achieved via HxTx-Hv1h inhibition on both CaV and KV channels of susceptible targets by blocking the channels directly, resulting in impediment of fundamental nervous system responses in neuronal, muscular, and cardiac functions. (Specific site of inhibition depends on the concentration of neurotoxin).

Toxicity Verification

HxTx-Hv1h is synthesized using the vector pTE28a-G1M5-His-SUMO-HxTx-Hv1h-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 HxTx-Hv1h 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-HxTx-Hv1h-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 HxTx-Hv1h to be highly toxic against T. urticae, achieving an fatality of 90.91% within 72 hours.

Fig2: (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 HxTx-Hv1h 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].

Fig7: 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