Before any design started......
Why choose E. coli Top10 as our chassis bacterial?
"Top10" is a commonly used strain of Escherichia coli with extensive literature support and operational experience. This makes it relatively easy to handle and maintain this strain in a laboratory, as researchers can typically access relevant experimental techniques and methods easily. In addition, Top10 strains usually have a highly stable genetic background, reducing unnecessary variations, which is crucial for precise experimental design.
The gene circuit we designed previously required the introduction of AND gate components into the chassis organism. In an AND gate, input signals must be present simultaneously to trigger an output signal, meaning there is no cross-interference between the inputs and the background. AND gates also need to produce reliable outputs when faced with various situations and conditions. Therefore, choosing the Top10 strain as the host organism helps ensure that our system exhibits orthogonality and robustness (as shown in Table 1 and Table 2).
Based on existing research, we ensured that the two sets of quorum-sensing elements do not interfere with the internal biological processes of the Top10 strain(Liu et al., 2018, Wang et al., 2011). This ensures that the genetic circuit is not affected by background noise, thereby enhancing the system's orthogonality.
In summary, the E. coli Top10 was chosen as the chassis organism for our project was based on its well-established track record, ease of handling, genetic stability, and compatibility with the specific requirements of our genetic circuits. This selection aligns with the principles of orthogonality and robustness.
Table 1. The best fits for the characterised responses of the cI/Plam based NOT gate using various RBSs in the selected context (E. coli MC1061, M9-glycerol, 30°C) (Wang et al., 2011)
Table 2. Chassis compatibility assays of the AND gate (Wang et al., 2011)
Why Choose Cysteine Protease Trypsin Inhibitor (CPTI) as a Protease Inhibitor?
The selection of CPTI as a protease inhibitor for our project was based on several key factors, each contributing to its suitability for our objectives. This choice aligns with the academic rigor required for our research and meets the criteria set by iGEM.
Successful Expression in E. coli:
First and foremost, CPTI has been successfully expressed in E. coli. In the screening and developing of protease inhibitors, priority is given to candidate molecules that have been successfully expressed in E. coli. This not only helps save time and resources but also increases the likelihood of success.
Bowman-Birk Family Classification:
Secondly, CPTI belongs to the Bowman-Birk family of protease inhibitors making the determination of its protease inhibitory activity relatively straightforward. This characteristic is crucial for monitoring and analyzing our work, ensuring that the selected variants or modifications possess the required protease inhibitory activity.
Broad-Spectrum Activity Against Pests:
Furthermore, CPTI exhibits a broad spectrum of activity in pest control, particularly against insects, making it suitable for inhibiting proteases in the gut of S. invicta. It has demonstrated significant inhibitory effects on various agricultural pests, making it a suitable candidate for targeting the digestive processes of insects that rely on serine proteases as their primary digestive enzymes. Importantly, CPTI has been shown to have minimal harmful effects on beneficial insects like bees (Zhu-jun et al., 2007).
Safety for Humans and Animals:
Last but not least, CPTI exhibits a high level of safety for humans and animals, which is a crucial consideration, ensuring that it does not harm non-target organisms during use. This contributes to reducing environmental risks and potential side effects. CPTI is known for its high safety profile for humans and animals. This consideration is of paramount importance in our research, ensuring that non-target organisms are not harmed during use. This not only reduces environmental risks but also mitigates potential side effects.
In conclusion, the selection of CPTI as a protease inhibitor for our project is grounded in its successful expression in E. coli, its Bowman-Birk family classification facilitating activity assessment, its broad-spectrum efficacy against pests, and its high safety standards for humans and animals. These factors collectively demonstrate its suitability for our research goals and align with the academic and iGEM standards.
Why Choose Cry3A-like Toxin as the Toxic Protein?
Following the suggestion from Dr. Daifeng Cheng, we conducted screenings from three major categories: plant lectins, neurotoxins, and parasporal crystals, to identify potential proteins with toxicity towards S. invicta (see human-practices). These screenings led us to three candidates: snowdrop lectin MGNA, Txp-Ⅰ neurotoxin, and parasporal crystals Cry3A-like toxin.
MGNA has been successfully expressed in E. coli BL21 (DE3) strain, but it is only toxic to piercing-sucking and certain chewing insects, and its toxicity to S. invicta is unclear(Longstaff et al., 1998, Martínez-Alarcón et al., 2018).
Txp-Ⅰ, originating from the bird mite, a parasite of membranous-winged insects, is a highly toxic neurotoxin to its hosts but has not undergone prokaryotic expression (Tomalski et al., 1989).
In comparison to these two toxins, the Cry3A-like toxin originates from a prokaryotic organism, a novel strain of Bacillus thuringiensis designated as UTD001. The native toxin protein has a size of 72.9 kDa, which, upon proteolytic cleavage by papain, forms an active protein of 66.6 kDa. This toxin has demonstrated clear toxicity to S. invicta in vitro (Carroll et al., 1997).
In our proof-of-concept validation, Cry3A-like toxin has been successfully expressed for extracellular secretion. Therefore, we have chosen Cry3A-like toxin as our toxic protein. (see Results section)
Objective 1: Enabling the Toxicity of Cry3A-like Toxin
We have successfully achieved extracellular secretion expression of the companion crystal Cry3A-like toxin (see Results section) . Additionally, there are substantial research results indicating that Cry3A-like toxin has demonstrated clear toxicity to S. invicta (Carroll et al., 1997).
Gastric toxicity experiments involve animal testing, which raises ethical and animal safety management considerations. Therefore, our initial concept is to utilize NCBI ( https://www.ncbi.nlm.nih.gov/) to screen for homologous receptors of Cry3A-like toxin known to exist in S. invicta. Subsequently, molecular docking will be employed to assess the protein-protein interaction capabilities of Cry3A-like toxin, thereby confirming its toxic effects (see Dry work).Objective 2: Eradicating S. invicta queen with the Toxic Protein
We analyzed and modeled the series of effects caused by the introduction of the drug protein into the S. invicta population. We constructed mathematical models in three key areas: in vivo expression of the drug, transmission and infection of the drug, and the transfer and accumulation of the drug. Based on the model simulation results, we discovered that the protease inhibitor CPTI and the Cry3A-like toxin from crystal cells exhibit a delayed but positive correlation in expression. Additionally, we discovered that the rate of drug protein enrichment within the ant queens is thousands of times higher than that in worker ants, allowing for precise eradication of S. invicta (see Model section for details)
Controlling the Low-Toxic Expression of Cry3A-like Toxin in Fourth-Instar Larvae
To ensure the continuous transfer of toxins from the fourth-instar larvae of S. invicta to the queen ants and throughout the entire ant colony, it is imperative that the toxin expressed within the fourth-instar larvae remains at low levels. Excessive toxin expression could lead to the premature death of the larvae, thereby disrupting this crucial pathway.
We have designed the following genetic circuit
1. Upon entering the larval gut, the anaerobic environment activates the pnirB promoter in the gut of engineered bacteria. This leads to the expression and secretion of CPTI, creating a favorable environment for the action of the toxic protein. Simultaneously, the first quorum sensing system, Tra, is initiated. As the bacterial density increases, the Tra quorum sensing system's signal molecules reach a threshold, it activates the expression of Cry3A-like toxin.
2. The engineered bacteria begin to release the toxic protein and initiate the second quorum sensing system, Las. Once the Las system's signal molecules reach a threshold, it triggers the expression of a lysis gene, causing cell lysis. Consequently, newly introduced engineered bacteria entering the gut will directly activate the lysis gene and cannot significantly express the toxic protein. This significantly reduces the accumulation rate of the toxic protein in the environment, creating a situation akin to a "threshold." This, in turn, achieves low-toxic expression of the drug within the larval gut.
3. Dry team conducted modeling of this process (link to model section).
The comprehensive process of QAA for eradicating S. invicta colonies can be outlined as follows:
1. A mixture of Top10 engineered bacteria deficient in essential nutrients and bait is distributed around the S. invicta nest. Worker ants forage for this bait and carry it back to the nest to feed the fourth-instar larvae of S.invicta.
2. The engineered bacteria colonize the larval gut, and as bacterial density increases, they first express and secrete CPTI that disrupts the gut proteases. This protects the Cry3A-like toxin. Subsequently, they undergo lysis, releasing Cry3A-like toxin. Due to quorum sensing signals in the environment reaching a threshold, the accumulation of all toxic proteins slows down after reaching a certain level. This delay postpones larval mortality.
3. Fourth-instar larvae of S. invicta, acting as the "digestive organs" for the entire colony, digest the proteins and spit predigested food. Multiple nurse ants feed this food to the queen, which requires protein for egg-laying. The Cry3A-like toxin accumulates in the queen's body through the feeding activity of multiple nurse ants, reaching a high dosage that ultimately causes the queen's death.
4. With the queen eliminated, the colony loses its capacity for further growth. The slow increase of CPTI and Cry3A-like toxin within the larval gut eventually kills the remaining larvae. Moreover, the engineered bacteria spread extensively within the colony through the food transfer pathway, hastening the colony's demise. This strategy dismantles the entire nest from its source.
Objective 3: Biosafety - Inducible Promoters and diaminopimelic acid (DAP) Nutrient Deficiency Strain
The ultimate goal of the biosafety switch experiment is to verify the effectiveness of the biosafety switch device (promoter + essential gene). Specifically, it aims to confirm whether our strain can grow under conditions similar to the S. invicta gut environment.
The gut of S. invicta is a low-oxygen environment; thus, we needed to select an anaerobic promoter. This anaerobic promoter must satisfy two conditions:
1) Under aerobic conditions, bacteria should not survive, so leaky expression of this anaerobic promoter in aerobic conditions should be as low as possible.
2) Under anaerobic conditions, bacteria should express a sufficient amount of essential growth growth factors. Therefore, the anaerobic promoter should have significantly higher expression under anaerobic conditions compared to aerobic conditions.
Fifteen oxygen-responsive promoters were experimentally compared, and six promoters meeting the two conditions mentioned above: nar-strong, nar-medium, nar-weak, nirB-medium, fnrF8, and yfiD-medium. Among these, the modified nirB (i.e., nirB-medium) exhibited the most significant difference in expression levels between anaerobic and aerobic conditions and was further characterized qualitatively. (Wichmann et al., 2023, Nasr and Akbari Eidgahi, 2014).
Figure 1. Characterization of oxygen-dependent promoter candidates using flow cytometry. (A) GFP fluorescence was normalized by subtracting fluorescence signals of the empty vector control. (B) Fold changes were calculated by dividing the normalized fluorescence signal obtained under anaerobic conditions by the normalized fluorescence signal obtained under aerobic conditions. Means (bars) and individual data points (dots) for n = 3 biologically independent samples are shown. Error bars indicate 95% confidence intervals (Wichmann et al., 2023).
Simultaneously, we knocked out the essential gene dapA in E. coli Top10, rendering it a diaminopimelic acid (DAP) synthesis-deficient strain wm3064. The dapA gene were ligated to the downstream of pnirB and cloned to pUC18T-mini-Tn7T plasmid, then transformed to wm3064. This device allows for dapA expression under anaerobic conditions, enabling bacterial growth (Dehio and Meyer, 1997). The genetically modified bacteria, after modification, will possess the following characteristics: they can only survive in with exogenously DAP supplement, such as the bait containing DAP or the anaerobic environment of the S. invicta gut. Once leaked into an aerobic environment without DAP, they cannot survival due to the nutritional deficiency.
Figure 2. The diagram of DAP deficient anaerobic strain was constructed by Red homologous recombination method
Objective 4 Establish a Multi-Insect Control Mechanism.
In insect genetic engineering research, pests can easy develop resistance to pesticide which target to a single gene. Currently, establishing multiple resistance mechanisms is an important and feasible measurement. By combining two proteins, Cry3A-like toxin and CPTI, which have completely different insecticidal mechanisms, the probability of insects developing resistance will be significantly reduced. This is because the likelihood of insects developing resistance is the product of the probabilities of developing resistance to each individual mechanism when they act together. (South and Hastings, 2018).