Initially through literature examinations, we have concluded a collection of mite venom peptides from predatory mites that exhibit strong extermination effects on spider mites via targeting calcium channels. All peptides are connected to GNA to achieve contact toxicity, giving BBa_K5184038, BBa_K5184042 , BBa_K5184043. In order to broaden the range of ion channels targetted, we included spider venom peptide in the collection. Thus, we obtained spider venom peptides BBa_K5184021,BBa_K5184032 ,BBa_K5184033 , targeting a variety of ion channels, including voltage-gated sodium channel, calcium ion activated potassium ion channels, and voltage-gated calcium channel. Then we also wanted to enhance the expression success rate of venom peptides, hence incorporating the G1M5-SUMO tag BBa_K5184022. Results show that all venom peptides exhibit strong exterminating effects on female Tetranychidae urticae, a representative of the Tetranychidae mites. We also added new toxicology data to this existing part,BBa_K1974001 , HxTx-hv1a, a venom peptide from funnel-web spider Hadronyche versus.

Part Numbers	Name	Type	Part Description
BBa_K1974001	Omega-hexatoxin-hv1a (adding new data)	Protein coding sequences	A venom peptide from the funnel-web spider, Hadronyche versus; targets insect CaV channels, leading to paralysis and death
BBa_K5184038	PpVP1-F	Protein coding sequences	MVP potentially targeting insect CaV channels, leading to paralysis and death.
BBa_K5184042	PpVP1-S	Protein coding sequences	MVP potentially targeting insect CaV channels, leading to paralysis and death.
BBa_K5184043	PpVP2-S	Protein coding sequences	MVP potentially targeting insect CaV channels, leading to paralysis and death.
BBa_K5184021	rCtx-4	Protein coding sequences	SVP targeting the insect NaV channels, leading to death and paralysis.
BBa_K5184032	Cs1A	Protein coding sequences	SVP targeting the insect CaV channels, leading to death and paralysis.
BBa_K5184033	HxTx-Hv1h	Protein coding sequences	SVP targeting the insect CaV and KCa channels, leading to death and paralysis.
BBa_K5184022	G1M5-SUMO-tag	Protein coding sequences	A recombinant tag that allows soluble expression of cysteine-rich venom peptides

To eliminate spider mites efficiently, safely, and specifically, we decided to seek out venom peptides originating from predatory mites. Our methods of locating the venom gene loci in mites will provide the iGEM community a feasible and efficient method of identifying potential mite venom peptides from genomic or transcriptomic data.

While the class of mites is very diverse in terms of their biology and appearance, their venom peptide gene proved to be highly conserved throughout evolution. It is therefore possible to locate venom loci in one mite species's genome or transcriptome using mRNA sequence or protein sequences of already identified mite venom peptides using BLAST tools.

In our project we located two P. persimilis venom peptides by BLASTing mRNA of two previously described N. barkeri venom peptides (GenBank OR995725.1 and OR995726.1) against the P. persimilis genome (GenBank assembly: GCA_037576195.1). Once the loci are identified [Fig1A], we aligned the BLAST results in the loci against the two barkeri mRNAs, thus obtaining coding and protein sequences of the persimilis venom peptides after filtering out the exons.

The venom peptides identified this way therefore contain signal peptides or other motifs that facilitate their secretion or production in vivo in their respective arachnid. For heterologous expression in unicellular chassis (e.g. E. coli, P. pastoris, etc.) however, the venom peptide can be truncated down to only its core venomous domain, a cysteine-rich domain characterized by a network of disulfide bridges that folds the peptide into a compact conformation. It is therefore possible to, via structural prediction results or sequence alignment results, match potential mite venom peptides with characterized core venomous domains of mite or spider venom peptides to identify the respective core venomous domains. In our project, we obtained structural prediction results of the persimilis and barkeri venoms using AlphaFold; matching their structures with (e.g. rCtx4 from [1]) [Fig1B], we truncated the venoms for efficient expression.

In order to overcome the difficulties encountered during attempts of using a single chassis for the synthesis of our terpene products, we have implemented a highly efficient fermentation system through co-culture of S. cerevisiae and E.coli, which provides the iGEM community with a novel and feasible method of co-culturing S. cerevisiae and E.coli. link protocol

1. Culture of E. coli: the E.coli transformants were streaked on LB plates with antibiotics and incubated at 37˚C. Then, tubes containing liquid LB with antibiotics were inoculated with fresh cell cultures of transformed E. coli and incubated overnight at 37 °C, 220rpm to obtain a seed solution. The E. coli seed solution is inoculated into the 2YT medium with antibiotics supplied with 1.5% glycerol at 37 °C, 220rpm. When OD600 reached 0.6−0.8, appropriate IPTG was added and the cultures were further incubated at 30 °C and 200 rpm for another 48h.


2. Culture of S. cerevisiae: the S. cerevisiae transformants were streaked on YPD+2% glucose plates and incubated at 30 ˚C. Then, tubes containing YPD medium supplemented with 2% glucose were inoculated with fresh cell cultures of a single colony of transformed S. cerevisiae and incubated overnight at 30 °C, 200rpm to obtain a seed solution. The S. cerevisiae seed solution is inoculated into the YPD medium supplied with 2% glucose for 24 hours at 30 °C and 200 rpm.


3. E. coli-S. cerevisiae Coculture: cultivated yeast cells were harvested by centrifugation at 6000×g for 5 min and then inoculate into E. coli cultures to make the final OD=40. Finally, a mixture of E. coli and S. cerevisiae was further cultured at 28 °C and 200 rpm for 48 h.

With the aim of expressing cysteine-rich venom peptides correctly, we have conducted a thorough investigation on plausible vectors that could fulfill this. We discovered the only feasible vector, being pET-G1M5-SUMO. This investigation hence has reference value in E.coli vector selection for expression of cysteine-rich peptides for the iGEM community.

Induced expression and SDS-PAGE analysis were carried out respectively after the construction of vectors, including pET-GNA-his, pET-his-GNA ,pET-PelB, pET-MalE, pET-PelB-SUMO, pET-MalE-SUMO, and pET-G1M5-SUMO. In which, only pET-G1M5-SUMO exhibit correct folding of venom peptides in the supernatant portion. Others are observed in the precipitate (P) after lysis, indicating inclusion bodies. Thus, the results suggest that only full secretion systems, such as G1M5, are capable of demonstrating correct expressions.

This investigation has reference value in selecting optimum protein expression condition for the iGEM community.

We conducted experiments under various culture conditions to optimize protein expression in E. coli. Protein expression conditions were applicable to all protein expressions in our project.

Initially, we attempted to induce HrpN protein expression under the following protocol:

1. Inoculate the culture until the OD600 reaches between 0.5-0.7.


2. Add 0.3 mM of IPTG for induction


3. Incubate the conical flask in a shaker 20°C after induction, 220 rpm for 16-20 hours.

However, this approach resulted in inclusion bodies [Fig. 1A].

We shortened the induction duration to 4 hours and temperature of incubation after induction was increased to 37°C. Results show that inclusion bodies significantly decreased as protein bands could be observed in the supernatant portion [Fig. 1B].

OD600 of induction was then optimized to 0.8-1.0 via 0.1 mM IPTG; subsequent temperature was lowered to 16 degrees Celcius. This resulted in a significant increase in the amount of HrpN protein present in the supernatant portion [Fig. 1C].