Engineering Success

After reviewing the literature, we discovered a synthetically engineered cyclic nonapeptide with specific toxicity towards S. invicta. This cyclic peptide can irreversibly alter the conformation of G-protein-coupled receptors (GPCRs) within the S. invicta, disrupting signal transduction pathways, ultimately leading to the death of S. invicta (Choi and Vander Meer, 2021). We cloned the nucleotide sequence encoding this peptide into the pET-28(b) vector using ΩPCR and fused it with multiple tags for expression.

However, upon completing the vector construction, we discovered the absence of a signal peptide sequence. Even if it successfully expressed, it could not assume the correct conformation. Given the limited available time and no literature support for the prokaryotic expression of this peptide, we deemed it imprudent to proceed with this work. Instead, the best course of action was to search for another suitable candidate protein. This candidate protein should not only exhibit toxicity to S. invicta but should also have literature support for successful expression in prokaryotes.

Through further literature research, we selected three additional toxin proteins: Snowdrop lectin (Galanthus nivalis agglutinin) GNA, neurotoxin Txp-I, and crystal protein Cry3A-like toxin.

While GNA has been successfully expressedin E. coli BL21 (DE3), previous reports suggest its toxicity is limited to piercing-sucking insects and some chewing insects, with unclear toxicity against S. invicta (Longstaff et al., 1998, Martínez-Alarcón et al., 2018, Khareedu et al., 1998, Gatehouse et al., 1984).

Txp-I neurotoxin is derived from the wheat curl mite, which parasitizes insects belonging to the Membracoidea and Heteroptera. It is highly toxic to its hosts but safe for mammals, however, it has not been expressed in prokaryotic suceefully (Tomalski et al., 1989, Tomalski and Miller, 1991).

In comparison to two other toxins, Cry3A-like toxin originates from prokaryotes, specifically a novel strain of Bacillus thuringiensis, UTD001. The original toxin has a size of 72.9 kDa, which, upon papain proteolytic cleavage in vitro, forms an active protein of 66.6 kDa with demonstrated clear toxicity against S. invicta (Carroll et al., 1997).

We screened for signal peptide OmpA-mediated secretion expression of the candidate proteins. Using Gibson Assembly, we cloned the “OmpA-candidate protein encoding sequence” into pET-30a, then transformed to E. coli BL21(DE3), with IPTG addition, only Cry3A-like toxin protein was successfully expressed and extracellular secreted.

Figure 1. SDS-PAGE Electrophoresis Detection of Cry3A-like Toxin Expression

Lane 1: Concentrated supernatant of pET-30a (+IPTG), Lane 2: Concentrated supernatant of pET30a-OmpA-Cry3A-like toxin (+IPTG), Lane 3: Concentrated supernatant of pET30a-OmpA-Cry3A-like toxin (-IPTG)

We will use enzymatic digestion/ΩPCR methods to add a 6×His tag to pET-30a-OmpA-Cry3A-like toxin. Subsequently, we will conduct western blot experiments to further confirm the secretion expression of Cry3A-like toxin.

The main problems encountered in this section are protein expression and whether the protein can be transported to extracellular. The modification process is mainly divided into three cycles.

In this phase, we attempted to express a trypsin inhibitor subtype A (TIA) protein with a ZnuA signal peptide. We selected this gene because previous research had already confirmed its successful expression in E. coli (Sultana et al., 2023), and it had shown a significant effect on S. invicta. However, after two months of attempts, we found that the TIA gene, whether with or without the ZnuA signal peptide, could not be expressed in E. coli. Therefore, we concluded that E. coli was not suitable for expressing the TIA gene, although previous research has reported the expression. Consequently, we had to select a new protease inhibitor, initiating the second cycle.

We decided to select a different protease inhibitor for our project, namely, Cowpea Trypsin Inhibitor (CPTI). CPTI is a plant-derived protein known for its broad-spectrum insecticidal properties. The reason for choosing this gene is reported that CPTI is successful expressed in E. coli (Zhu-jun et al., 2007, Yang et al., 2003). This provided us with confidence in achieving successful protein expression.

In contrast to the original literature, we opted not to add a GST purification tag to the CPTI gene. Our goal was to use CPTI directly for the eradication of S. invicta, rather than purifying the protein and subsequently removing the GST tag. Notably, the CPTI protein, when devoid of the GST tag, processed a molecular weight of only 10.4 kDa. As a result, 12% SDS-PAGE was not sufficient to visualize the protein effectively. To address this, Tricine-PAGE and SDS-PAGE with 17.5% acrylamide were conducted.

However, the results indicated that the CPTI gene without the GST tag could not express properly, as previous report. Consequently, we decided to follow the literature and add the GST tag to the vector, starting the third cycle.

We successfully add the GST tag to the CPTI gene, and protein expression induction is currently in progress. This modification ensures that the protein can be expressed to a significant extent. Literature has previously demonstrated the successful expression of CPTI with the GST tag (Yang et al., 2003). Our unique approach in this cycle is to assess the activity of CPTI without removing the GST tag.

Initially, we set the project goal to modify a symbiotic bacterium inside S. invicta to secrete toxins for eradicating them. The experimental strategy was designed as follows: the toxin protein was continuous expressed within the engineered bacteria, when the population of the engineered bacteria reach a certain concentration in the ant's gut, the lysis genes will be activated, allowing the toxin protein to rapidly reach high concentrations, effectively exterminating the ants.

However, through the study of S. invicta behavior and expert interviews, we learned that S. invicta are highly social organisms. (see human-practices) Their food-sharing mechanisms, coupled with the queen's prolific reproduction and high toxicity, hinder the spread of toxins within the ant colony. Rapidly and efficiently killing a portion of ants within a short time could not completely destroy the ant colony and would deter ants from consuming bait containing the engineered bacteria.

Based on this understanding, the release mechanism of the toxin protein was modified from lysis to secretion, allowing the engineered bacteria to maintain continuous expression of the toxin protein. Since food passes through the digestion of fourth-instar larvae first, we introduced protease inhibitors and two orthogonal quorum-sensing systems. This ensured that the protease inhibitor was released into the gut first, protecting the subsequent activity of the toxin protein. To control the expression level of the toxin protein, we utilized a binary oscillation system with negative feedback, which allowed the expression of the toxin protein to inhibit the expression of upstream genes, reducing the expression of the toxin protein itself and thereby stabilizing the expression levels of both the protease inhibitor and the toxin protein. The Dry team also demonstrated the feasibility of oscillatory expression through mathematical modeling. However, in the subsequent experiment design, we discovered that the oscillatory pathway itself posed significant complexity in adjusting various parameters of the genetic circuit. Moreover, the internal environment within S. invicta is highly intricate and challenging to predict, making it potentially difficult to replicate the results obtained in the laboratory. This May when we attend the iGEM Southern China Regional Meeting, experienced iGEM advisors pointed out that our oscillatory pathway might not be the most optimal approach to achieve our intended goals. (see human-practices)

Ultimately, we quite the intriguing concept of oscillatory expression and optimized the pathway to its final version. The oscillation-sustaining module was removed and lysis genes was introduced. When the quorum-sensing signal reaches the threshold, the engineered bacteria start lysing and terminate product expression. We use the quorum-sensing threshold to control the maximum expression level of the product. Through food-sharing within the ant colony, the toxin protein will be disseminated throughout the entire colony and accumulates within the queen's body, achieving the complete eradication of S. invicta society.

Firstly, the pnirB promoter was planned to digest and ligate to the promoterless eGFP in the plasmid of pET-28b-singal-eGFP. Then the difference in promoter expression strength was measured by the fluorescence intensity under aerobic or anaerobic environments.

However, multiple attempts was conducted, but the target plasmid was not obtained. The short length of the pnirB promoter fragment (78 bp after adding the ribosome binding site) may have led to a relatively low success rate in digestion and ligation. After several unsuccessful attempts, ΩPCR was employed to construct the plasmid. Ultimately, the pnirB was firstly added to eGFP by overlap PCR, then the whole fragment was ligated to pET-28b.

We try to measure the strength of promoter expression under aerobic or anaerobic environments by the fluorescence intensity of eGFP. However, another report showed the eGFP could not work at anaerobic environment. Therefore, eGFP was replaced with a gentamicin (Gm) resistance gene by the same method. The promoter's activity was validated by the growth performance of strain containing the plasmid pET-28b-pnirB-Gmr under aerobic or anaerobic with gradient Gm concentrations.

In addition to the anaerobic promoter, we explored methods for constructing auxotrophic strains to enhance the feasibility of our biosafety device.

The fabA was linked to the anaerobic promoter pnirB and then inserted into the genome to replace the original fabA gene in the chromosome by λ Red recombination. So that, the engineered bacteria could only survive by expressing fabA in the anaerobic environment or with the exogenous oleic acid. Several attempts had been conducted, but no colony was obtained with the oleic acid addition. One possible reason is the fabA is the essential gene for E.coil, it may not only important in the unsaturated fatty acid synthesis, but also important to other metabolism. Another thought is that the regulation of fabA is vital to determine the ratio of saturated fatty acid and unsaturated fatty acid, because the downstream gene fabB was not only play a role in the unsaturated fatty acid synthesis but also in the saturated fatty acid synthesis.

pnirB-dapA was cloned into the pUC18T-mini Tn7T plasmid and then transformed to mw3064 (dapA mutant strain). The growth of resulting strain is strictly dependent on the supply of exogenous DAP or DAP expressed in low-oxygen environment. The growth of dapA- pUC18T-mini-Tn7T/△dapA and △dapA was detected under aerobic and anaerobic conditions, respectively. The results showed that △dapA could only grow under anaerobic and aerobic conditions with the addition of DAP. And dapA- pUC18T-mini-Tn7T/△dapA can grow under both conditions.

The dapA gene in the Top10 strain will be knockout to make the safety device stable in expression. In addition, the random mutation to the sequence of pnirB or the RBS will be conduct to further improve the accuracy of pnirB expression between the anaerobic and aerobic condition.