Difference between revisions of "Part:BBa K5184038"

Latest revision as of 13:51, 12 November 2024

PpVP1-F

Essential Information

Abstract

In our project we employ venom peptides to act as insecticidal agents against spider mites. This part presents an original mite venom peptide with high potency against spider mites that is determined by in vivo testing against T. urticae. This mite venom peptide may also aid future iGEM teams in identifying homologous venom peptide genes in the acariforme superorder or even across the acarid subclass that is right now almost completely unexplored.

The part collection includes: Parts expressing venom peptides that target various ion channels that leads to paralysis and death of spider mites: PpVP1S (BBa_K5184042), PpVP2S (BBa_K5184043), PpVP1F (BBa_K5184038), rCtx-4 (BBa_K5184021), HxTx-Hv1h (BBa_K5184033), Cs1A (BBa_K5184032); supplementary parts that allows extracellular secretion, facilitates venom peptide folding, and improves contact and oral toxicity (BBa_5184020, and BBa_K5184022); composite parts that guarantee correct folding of venom peptides exhibiting contact toxicity (BBa_K5184071-BBa_K5184076).

Our part collection provides a feasible and efficient expression of a collection of venom peptides targeting a diverse range of molecular targets in spider mites. While any one of the venom peptides in the collection can achieve reasonable elimination efficiency, combination of multiple venom peptides with different molecular targets makes resistance development much harder. This can help and inspire future teams to design and perfect venom peptide-based pesticides that are capable of tackling many pests known for their prowness for developing resistance and, as template sequences for identification of other acariforme venom peptides.

Basic Characterization

PpVP1F is the modified version of the venom peptide we identified in genome of the predatory mite, Phytoseiulus persimilis, by genome mining. It shares a high degree of homology to venom peptides from the predatory mite species Neoseiulus barkeri, which is first identified in [1]. Via phylogenic analysis, we believe it likely achieves its paralyzing and insecticidal properties by interfering with voltage gated calcium channels whose affinity towards is then verified by computer modelling.

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]

Characterization

Molecular Structure

PpVP1F, composing of a total of 74 amino acids, contains 11 cysteine residues, 8 of which involved in forming 4 disulfide bridges, as well as two antiparallel beta sheets, together contributing to the final highly compact conformation of the venom. Comparing to NbVP1[1], it has the same set of cysteine network of C1xxxC2xxxC3C4xxxC5xC6xxxC7xC8, with disulfide bridges between C(1)C(4), C(2)C(5), C(3)C(8), and C(6)C(7)[Fig.1A], and therefore shares an almost congruent geometry [Fig.1B], differing by only 5 amino acids.

Fig.1: A. Disulfide network of the venomous domain of PpVP2S, involving 8 cysteine residues and 4 disulfide bridges B. Comparison of structural prediction results of core venomous domains of NbVP1F (transparent light gray) and PpVP1F (solid blue) from AlphaFold Server; the cysteine residues are colored orange

Genome Mining

Using identified mRNAs of the two N. barkeri venom peptides as query, we identified the two orthologous venom peptide-encoding loci in the P. persimilis genome (GenBank Assembly: GCA_037576195.1) using NCBI BLASTn [Fig.2]. Sequences from the BLAST result are then aligned with the two N. barkeri mRNAs to filter out the introns. Thus, the protein sequence of the P. persimilis orthologous venom peptides are obtained.

Fig.2: BLAST results of two N. barkeri venom peptide mRNAs against the P. persimilis genome.

in silico Analysis

With further research into spider venom peptides’ molecular mechanisms for their paralyzing and lethal effects, we realized that they (and therefore MVPs due to high levels of homology between them) target neuronal ion channels or receptors. Their interference led to blockage of various receptors or ion channels or neurotransmitter receptors, leading to prevention of release or detection of neurotransmitters. This therefore interferes with normal conduction of neural impulses and achieves paralyzing and lethal effects. To analyze molecular action of venom peptides using computational approaches such as molecular docking, we constructed a phylogenic tree based on core venomous domains of various SVPs with known molecular targets [3], [4], [5], [6] along with the four MVPs, in an attempt to determine their molecular targets. The results, in [Fig4], suggests that they likely interfere with voltage-gated calcium channels. We therefore tried to predict molecular effects of the venom peptides against the CaV channels, using both “conventional” docking tools such as HDock server, and “non-classical” tools such as AlphaFold (for its ability to accurately predict conformational changes in both receptor and a peptide ligand upon binding). The docking results from both AlphaFold and HDock suggests that the MVPs are likely to interfere with the alpha 2 subunit of the T. urticae CaV channels, blocking flow of ions and therefore release of neurotransmitters.

Fig.4: A. Phylogenic tree from core venomous domains of MVPs and several SVPs with known molecular targets (all ion channels, ion type identified at the end of each name) B. Docking results of PpVP1F's core venmous domain against T. urticae voltage-gated calcium channel using AlphaFold

Toxicity Assay

The designed and optimized venom peptides are expressed using the expression system G1M5-His-SUMO-MVP-GNA, where G1M5-SUMO proved to facilitate correct folding and therefore soluble expression of the attached protein [7][Fig.5A], and GNA, which enhances the fusion protein’s oral and contact toxicity [8]. Plasmids pET28a-G1M5-His-SUMO-MVP-GNA [Fig.5B] are assembled using GoldenGate clonining, and is transformed into BL21(DE3) strain. After IPTG induction and overnight incubation, the culture is harvested and underwent cell lysis; SDS-PAGE is then ran for supernatant and precipitate. The supernatant is then treated with SUMO protease [Fig.5B].

Fig5: A. G1M5 signal peptide meditates extracellular secretion of the fusion protein B. Plasmid construct pET28a-G1M5-His-SUMO-PpVP1F-GNA-His C. SDS-PAGE of SUMO-digested supernatant of PpVP1F, with supernatant of BL21(DE3) as control; S: supernatant, SUMO: SUMO protease-treated supernatant

Supernatant from cell lysis is treated with SUMO protease to, ideally, give MVP-GNA fusion proteins. For toxicity assay, Professor Huang of SCAU tested the contact toxicity of SUMO-protease-treated supernatant on 3 groups of 20 females of Tetranychus urticae, which is the most prominent specie in spider mites [14]. The treated supernatant is applied to the spider mites using a spraying method, with supernatant of induced BL21(DE3) that underwent the same treatments as control. The results reveal that PpVP2S shows very high toxicity against T. urticae [Fig.6C&D], achieving a death rate of 98.25% in the first day and 100% in the next.

Fig6: A. T. urticae before being sprayed with PpVP1F B. T. urticae after being sprayed with PpVP1F C. Survival plot of PpVP1F 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 PpVP1F 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

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

References

[1]: Chen, L., Adang, M. J., & Shen, G. (2024c). A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests. Pesticide Biochemistry and Physiology, 202, 105963. https://doi.org/10.1016/j.pestbp.2024.105963
[2]: 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
[3]: Wu, X.; Chen, Y.; Liu, H.; Kong, X.; Liang, X.; Zhang, Y.; Tang, C.; Liu, Z. The Molecular Composition of Peptide Toxins in the Venom of Spider Lycosa coelestis as Revealed by cDNA Library and Transcriptomic Sequencing. Toxins 2023, 15, 143. https://doi.org/10.3390/toxins15020143
[4]: Ahmed, J.; Walker, A.A.; Perdomo, H.D.; Guo, S.; Nixon, S.A.; Vetter, I.; Okoh, H.I.; Shehu, D.M.; Shuaibu, M.N.; Ndams, I.S.; et al. TwoNovel Mosquitocidal Peptides Isolated from the Venom of the Bahia Scarlet Tarantula (Lasiodora klugi). Toxins 2023, 15, 418. https://doi.org/10.3390/toxins15070418
[5]: Ho, T. N., Turner, A., Pham, S. H., Nguyen, H. T., Nguyen, L. T., Nguyen, L. T., & Dang, T. T. (2023). Cysteine-rich peptides: From bioactivity to bioinsecticide applications. Toxicon, 230, 107173. https://doi.org/10.1016/j.toxicon.2023.107173
[6]: Guo, R.; Guo, G.; Wang, A.; Xu, G.; Lai, R.; Jin, H. Spider-Venom Peptides: Structure, Bioactivity, Strategy, and Research Applications. Molecules 2024, 29, 35. https://doi.org/10.3390/molecules29010035
[7]: Shad, M., Nazir, A., Usman, M., Akhtar, M. W., & Sajjad, M. (2024b). Investigating the effect of SUMO fusion on solubility and stability of amylase-catalytic domain from Pyrococcus abyssi. International Journal of Biological Macromolecules, 266, 131310. https://doi.org/10.1016/j.ijbiomac.2024.131310
[8]: Fitches EC, Pyati P, King GF, Gatehouse JA. Fusion to snowdrop lectin magnifies the oral activity of insecticidal ω-Hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system. PLoS One. 2012;7(6):e39389. doi: 10.1371/journal.pone.0039389. Epub 2012 Jun 22.
[9]: Windley, M. J., Herzig, V., Dziemborowicz, S. A., Hardy, M. C., King, G. F., & Nicholson, G. M. (2012b). Spider-Venom peptides as bioinsecticides. Toxins, 4(3), 191–227. https://doi.org/10.3390/toxins4030191

@@ Line 5: / Line 5: @@
 ==Essential Information==
 ===Abstract===
-In our project we employ venom peptides to act as insecticidal agents against spider mites. This part presents an original mite venom peptide with high potency against spider mites that is determined by in vivo testing against ''T. urticae''. This mite venom peptide may also aid future iGEM teams in identifying homologous venom peptide genes in the acariforme superorder or even across the acarid subclass that is right now almost completely unexplored.
+In our project we employ venom peptides to act as insecticidal agents against spider mites. This part presents an original mite venom peptide with high potency against spider mites that is determined by in vivo testing against ''T. urticae''. This mite venom peptide may also aid future iGEM teams in identifying homologous venom peptide genes in the acariforme superorder or even across the acarid subclass that is right now almost completely unexplored. <br><br>
-The part collection includes: Parts expressing venom peptides that target various ion channels that leads to paralysis and death of spider mites: PpVP1S (BBa_K5184042), PpVP2S (BBa_K5184043), PpVP1F (BBa_K5184038), rCtx-4 (BBa_K5184021), HxTx-Hv1h (BBa_K5184033), Cs1A (BBa_K5184032); supplementary parts that allows extracellular secretion, facilitates venom peptide folding, and improves contact and oral toxicity (BBa_5184020, and BBa_K5184022); composite parts that guarantee correct folding of venom peptides exhibiting contact toxicity (BBa_K5184071-BBa_K5184076).
+The part collection includes: Parts expressing venom peptides that target various ion channels that leads to paralysis and death of spider mites: PpVP1S (BBa_K5184042), PpVP2S (BBa_K5184043), PpVP1F (BBa_K5184038), rCtx-4 (BBa_K5184021), HxTx-Hv1h (BBa_K5184033), Cs1A (BBa_K5184032); supplementary parts that allows extracellular secretion, facilitates venom peptide folding, and improves contact and oral toxicity (BBa_5184020, and BBa_K5184022); composite parts that guarantee correct folding of venom peptides exhibiting contact toxicity (BBa_K5184071-BBa_K5184076). <br><br>
 Our part collection provides a feasible and efficient expression of a collection of venom peptides targeting a diverse range of molecular targets in spider mites. While any one of the venom peptides in the collection can achieve reasonable elimination efficiency, combination of multiple venom peptides with different molecular targets makes resistance development much harder. This can help and inspire future teams to design and perfect venom peptide-based pesticides that are capable of tackling many pests known for their prowness for developing resistance and, as template sequences for identification of other acariforme venom peptides.
 ===Basic Characterization===
-PpVP2S is the modified version of the venom peptide we identified in genome of the predatory mite, ''Phytoseiulus persimilis'', by genome mining. It shares a high degree of homology to venom peptides from the predatory mite species ''Neoseiulus barkeri'', which is first identified in [1]. Via phylogenic analysis, we believe it likely achieves its paralyzing and insecticidal properties by interfering with voltage gated calcium channels whose affinity towards is then verified by computer modelling.
+PpVP1F is the modified version of the venom peptide we identified in genome of the predatory mite, ''Phytoseiulus persimilis'', by genome mining. It shares a high degree of homology to venom peptides from the predatory mite species ''Neoseiulus barkeri'', which is first identified in [1]. Via phylogenic analysis, we believe it likely achieves its paralyzing and insecticidal properties by interfering with voltage gated calcium channels whose affinity towards is then verified by computer modelling.
 ===Sequences===
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K5184043 SequenceAndFeatures</partinfo>
+<partinfo>BBa_K5184038 SequenceAndFeatures</partinfo>
 ==Characterization==
 ===Molecular Structure===
-PpVP2S is the truncated version of the PpVP2F, composing of a total of 55 amino acids, containing 8 cysteine residues that form 4 disulfide bridges, and two antiparallel beta sheets, together contributing to the final highly compact conformation of the venom. Comparing to NbVP2[1], it has the same set of cysteine network of C1xxxC2xxxC3C4xxxC5xC6xxxC7xC8, with disulfide bridges between C(1)C(4), C(2)C(5), C(3)C(8), and C(6)C(7)[Fig.1A], and therefore shares an almost congruent geometry [Fig.1B], differing by only 5 amino acids.
+PpVP1F, composing of a total of 74 amino acids, contains 11 cysteine residues, 8 of which involved in forming 4 disulfide bridges, as well as two antiparallel beta sheets, together contributing to the final highly compact conformation of the venom. Comparing to NbVP1[1], it has the same set of cysteine network of C1xxxC2xxxC3C4xxxC5xC6xxxC7xC8, with disulfide bridges between C(1)C(4), C(2)C(5), C(3)C(8), and C(6)C(7)[Fig.1A], and therefore shares an almost congruent geometry [Fig.1B], differing by only 5 amino acids.
-<center><html><img src="https://static.igem.wiki/teams/5184/parts/mvp1.webp" width="600"/></html></center>
+<center><html><img src="https://static.igem.wiki/teams/5184/parts/p1mvp1.webp" width="600"/></html></center>
-<center><b>Fig.1: A. Disulfide network of the venomous domain of PpVP2S, involving 8 cysteine residues and 4 disulfide bridges B. Comparison of structural prediction results of core venomous domains of NbVP2F (transparent light gray) and PpVP2F (solid blue) from AlphaFold Server; the cysteine residues are colored orange</b></center>
+<center><b>Fig.1: A. Disulfide network of the venomous domain of PpVP2S, involving 8 cysteine residues and 4 disulfide bridges B. Comparison of structural prediction results of core venomous domains of NbVP1F (transparent light gray) and PpVP1F (solid blue) from AlphaFold Server; the cysteine residues are colored orange</b></center>
 ===Genome Mining===
 Using identified mRNAs of the two ''N. barkeri'' venom peptides as query, we identified the two orthologous venom peptide-encoding loci in the ''P. persimilis'' genome (GenBank Assembly: GCA_037576195.1) using NCBI BLASTn [Fig.2].
@@ Line 24: / Line 24: @@
 <center><html><img src="https://static.igem.wiki/teams/5184/parts/mvp2.webp" width="600"/></html></center>
 <center><b>Fig.2: BLAST results of two ''N. barkeri'' venom peptide mRNAs against the ''P. persimilis genome''.</b></center>
-===Truncation & Optimizations===
-Looking into molecular features of the sequences of the mite venom peptides: small, cysteine-rich and involves multiple disulfide bridges, we realized the mite venom peptides to be homologous to spider venom peptides, a class of versatile, widely-studied venom peptides found in spiders—phylogenically related to the acariformes.
-With some research into spider venom peptides, we realized the cysteine-rich venom peptides’ toxicity is largely determined by a core venomous domain, and that other domains are largely irrelevant to the inhibitory effects the venom has on its molecular targets (illustrated dramatically by the fact that many recombinant SVPs retain only their core venomous domain, yet retains and usually achieves toxicity higher than that of their native forms).
-Since the mite venom peptide protein sequences are obtained directly from the genome of ''P. persimilis'', the protein likely include various domains or structures that facilitates the protein’s expression and post-translational modifications and secretion in vivo. Since they are largely unnecessary for heterologous expression in unicellular chassis such as E. coli or P. Pastoris, such regions, could be truncated to improve expression efficiency and toxicity.
-We therefore, matching structural prediction results of the MVPs and a described, recombinant SVP (truncated down to only the central venomous domain), rCtx4 [2], identified the MVP’s core venom domain.
-<center><html><img src="https://static.igem.wiki/teams/5184/parts/p2f-rctx4-superimposed.webp" width="600"/></html></center>
-<center><b>Fig.3: Structural comparisons between AlphaFold Structural prediction results of PpVP2F (White and blue), and rCtx4 (transparent light grey); the identified core venomous domain of PpVP2F is colored in blue and the cysteine resideus colored orange</b></center>
 ===''in silico'' Analysis===
 With further research into spider venom peptides’ molecular mechanisms for their paralyzing and lethal effects, we realized that they (and therefore MVPs due to high levels of homology between them) target neuronal ion channels or receptors. Their interference led to blockage of various receptors or ion channels or neurotransmitter receptors, leading to prevention of release or detection of neurotransmitters. This therefore interferes with normal conduction of neural impulses and achieves paralyzing and lethal effects.
@@ Line 36: / Line 29: @@
 We therefore tried to predict molecular effects of the venom peptides against the CaV channels, using both “conventional” docking tools such as HDock server, and “non-classical” tools such as AlphaFold (for its ability to accurately predict conformational changes in both receptor and a peptide ligand upon binding).
 The docking results from both AlphaFold and HDock suggests that the MVPs are likely to interfere with the alpha 2 subunit of the ''T. urticae'' CaV channels, blocking flow of ions and therefore release of neurotransmitters.
-<center><html><img src="https://static.igem.wiki/teams/5184/parts/mvp3.webp" width="600"/></html></center>
+<center><html><img src="https://static.igem.wiki/teams/5184/parts/p1mvp3.webp" width="600"/></html></center>
-<center><b>Fig.4: A. Phylogenic tree from core venomous domains of MVPs and several SVPs with known molecular targets (all ion channels, ion type identified at the end of each name) B. Docking results of PpVP2S against T. urticae voltage-gated calcium channel using AlphaFold</b></center>
+<center><b>Fig.4: A. Phylogenic tree from core venomous domains of MVPs and several SVPs with known molecular targets (all ion channels, ion type identified at the end of each name) B. Docking results of PpVP1F's core venmous domain against T. urticae voltage-gated calcium channel using AlphaFold</b></center>
 ===Toxicity Assay===
 The designed and optimized venom peptides are expressed using the expression system G1M5-His-SUMO-MVP-GNA, where G1M5-SUMO proved to facilitate correct folding and therefore soluble expression of the attached protein [7][Fig.5A], and GNA, which enhances the fusion protein’s oral and contact toxicity [8].
-Plasmids pET28a-G1M5-His-SUMO-MVP-GNA [Fig.5B] are assembled using GoldenGate clonining, and is transformed into BL21(DE3) strain. After IPTG induction and overnight incubation, the culture is harvested and underwent cell lysis; SDS-PAGE then ran for supernatant and precipitate. The supernatant is then treated with SUMO protease [Fig.5B].
+Plasmids pET28a-G1M5-His-SUMO-MVP-GNA [Fig.5B] are assembled using GoldenGate clonining, and is transformed into BL21(DE3) strain. After IPTG induction and overnight incubation, the culture is harvested and underwent cell lysis; SDS-PAGE is then ran for supernatant and precipitate. The supernatant is then treated with SUMO protease [Fig.5B].
-<center><html><img src="https://static.igem.wiki/teams/5184/parts/ppvp2s-sumo.webp" width="600"/></html></center>
+<center><html><img src="https://static.igem.wiki/teams/5184/parts/ppvp1f-sumo.webp" width="600"/></html></center>
-<center><b>Fig5: A. G1M5 signal peptide meditates extracellular secretion of the fusion protein B. Plasmid construct pET28a-G1M5-His-SUMO-PpVP2S-GNA-His C. SDS-PAGE of SUMO-digested supernatant of PpVP2S, with supernatant of BL21(DE3) as control; S: supernatant, SUMO: SUMO protease-treated supernatant</b></center>
+<center><b>Fig5: A. G1M5 signal peptide meditates extracellular secretion of the fusion protein B. Plasmid construct pET28a-G1M5-His-SUMO-PpVP1F-GNA-His C. SDS-PAGE of SUMO-digested supernatant of PpVP1F, with supernatant of BL21(DE3) as control; S: supernatant, SUMO: SUMO protease-treated supernatant</b></center>
 Supernatant from cell lysis is treated with SUMO protease to, ideally, give MVP-GNA fusion proteins. For toxicity assay, Professor Huang of SCAU tested the contact toxicity of SUMO-protease-treated supernatant on 3 groups of 20 females of ''Tetranychus urticae'', which is the most prominent specie in spider mites [14]. The treated supernatant is applied to the spider mites using a spraying method, with supernatant of induced BL21(DE3) that underwent the same treatments as control. The results reveal that PpVP2S shows very high toxicity against ''T. urticae'' [Fig.6C&D], achieving a death rate of 98.25% in the first day and 100% in the next.
-<center><html><img src="https://static.igem.wiki/teams/5184/parts/ppvp2s-lethality.webp" width="600"/></html></center>
+<center><html><img src="https://static.igem.wiki/teams/5184/parts/p1f-lethality1.webp" width="600"/></html></center>
-<center><b>Fig6: A. ''T. urticae'' before being sprayed with PpVP2S B. ''T. urticae'' after being sprayed with PpVP2S C. Survival plot of PpVP2S 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 PpVP2S 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>
+<center><b>Fig6: A. ''T. urticae'' before being sprayed with PpVP1F B. ''T. urticae'' after being sprayed with PpVP1F C. Survival plot of PpVP1F 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 PpVP1F 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>
 ==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].
-<center><html><img src="https://static.igem.wiki/teams/5184/parts/vp-lethality.webp" width="600"/></html></center>
+<center><html><img src="https://static.igem.wiki/teams/5184/parts/vp-lethality-v.webp" width="600"/></html></center>
 <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>
@@ Line 56: / Line 49: @@
 !Current VP!!Venom Name!!Targeted Ion Channel!!New?!!Part Number!!Original Specie
 |-
-|✳️||PpVP2S||Ca||New||BBa_K5184043||''Phytoseiulus persimilis''
+|||PpVP2S||Ca||New||BBa_K5184043||''Phytoseiulus persimilis''
 |-
 |||PpVP1S||Ca||New||BBa_K5184042||''Phytoseiulus persimilis''
 |-
-|||PpVP1F||Ca||New||BBa_K5184038||''Phytoseiulus persimilis''
+|✳️||PpVP1F||Ca||New||BBa_K5184038||''Phytoseiulus persimilis''
 |-
 |||rCtx4||Na||||BBa_K5184021||''Phoneutria depilata''
@@ Line 68: / Line 61: @@
 |||HxTx-Hv1h||Ca, K||||BBa_K5184033||''Hadronyche versuta''
 |}
+===References===
+[1]: Chen, L., Adang, M. J., & Shen, G. (2024c). A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests. Pesticide Biochemistry and Physiology, 202, 105963. https://doi.org/10.1016/j.pestbp.2024.105963 <br>
+[2]: 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 <br>
+[3]: Wu, X.; Chen, Y.; Liu, H.;  Kong, X.; Liang, X.; Zhang, Y.; Tang,  C.; Liu, Z. The Molecular  Composition of Peptide Toxins in the  Venom of Spider Lycosa coelestis as  Revealed by cDNA Library and  Transcriptomic Sequencing. Toxins  2023, 15, 143. https://doi.org/10.3390/toxins15020143 <br>
+[4]: Ahmed, J.; Walker, A.A.;  Perdomo, H.D.; Guo, S.; Nixon, S.A.;  Vetter, I.; Okoh, H.I.; Shehu, D.M.;  Shuaibu, M.N.; Ndams, I.S.; et al.  TwoNovel Mosquitocidal Peptides  Isolated from the Venom of the Bahia  Scarlet Tarantula (Lasiodora klugi).  Toxins 2023, 15, 418. https://doi.org/10.3390/toxins15070418 <br>
+[5]: Ho, T. N., Turner, A., Pham, S. H., Nguyen, H. T., Nguyen, L. T., Nguyen, L. T., & Dang, T. T. (2023). Cysteine-rich peptides: From bioactivity to bioinsecticide applications. Toxicon, 230, 107173. https://doi.org/10.1016/j.toxicon.2023.107173 <br>
+[6]: Guo, R.; Guo, G.; Wang, A.;  Xu, G.; Lai, R.; Jin, H. Spider-Venom  Peptides: Structure, Bioactivity,  Strategy, and Research Applications.  Molecules 2024, 29, 35. https://doi.org/10.3390/molecules29010035 <br>
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 ===Functional Parameters===
 <partinfo>BBa_K5184043 parameters</partinfo>
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