Difference between revisions of "Part:BBa K5184021"

Revision as of 03:51, 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 other venom peptides target. 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.

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

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]

[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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 <partinfo>BBa_K5184021 short</partinfo>
-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.
+In order to eliminate spider mites, the spider venom peptide rCtx-4 is incorporated in our project to broaden the spectrum of molecular targets other venom peptides target. 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''.
 ===Usage and Biology===
-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.
+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.
+<center><html><img src="https://static.igem.wiki/teams/5184/parts/rctx4-structure.webp" width="600"/></html></center>
+<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>
+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===
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
@@ Line 12: / Line 17: @@
+[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
 <!-- Uncomment this to enable Functional Parameter display
 ===Functional Parameters===
 <partinfo>BBa_K5184021 parameters</partinfo>
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