Difference between revisions of "Part:BBa K5184021"

Revision as of 11:07, 27 September 2024

rCtx-4

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.

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. 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]

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 <partinfo>BBa_K5184021 short</partinfo>
-"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.
+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 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."
-<!-- Add more about the biology of this part here
 ===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.
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 <span class='h3bb'>Sequence and Features</span>