Plasmid construction
Figure 1. Diagram of Cry3A-like Toxin circuit design
To verify the secretory expression of Cry3A-like toxin and its function, we performed the following experiments:
The BL21 (DE3) strain was cultured in LB broth overnight, and on the next day, the culture was transferred to fresh LB and incubated at 37℃ till the OD600 reach 0.6, then IPTG was added and induced for 3 h. Then the organisms and supernatant were separated by spinning in a centrifuge at 6,000 rpm for 10 min. The expression results were checked by SDS-PAGE electrophoresis method. If the target bands were detected in the electrophoresis results of the supernatant protein extracts, the Cry3A-like toxin was successfully secreted and expressed.
Gastric toxicity experiments necessitate animal testing, which gives rise to ethical and animal safety concerns. Therefore, our initial concept is to utilize NCBI (https://www.ncbi.nlm.nih.gov/) to search for receptors with similarities to the Cry3A-like toxin, which is known to be present 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).
Plasmid construction
Figure 2. Diagram of CPTI circuit design
To confirm whether CPTI is expressed and its function, we performed the following experiments.
The protein was prepared as described above. The precipitate and the supernatant were then separated by centrifugation with the speed of 12,000 rpm for 10 min. The supernatant was concentrated by ultrafiltration centrifuge tubes, while the precipitate was treated with lysis buffer and lysed by sonication. The expression results were obtained by Tricine-PAGE assay and Western Blot assay.
We intend to determine the activity of the expressed protease inhibitor using the BAEE (Na-Benzoyl-L-arginine ethyl ester) assay, which is based on the principle outlined below:
The BAEE assay, used to measure trypsin inhibitor (TI) activity, relies on the ability of trypsin inhibitors to bind to trypsin. BAEE serves as a substrate catalyzed by trypsin, and the product of trypsin-catalyzed BAEE hydrolysis absorbs light at a wavelength of 253 nm. The rate of increase in A253 (absorbance at 253 nm) per unit time (min) corresponds to the activity of trypsin (U1). When TI is added to the reaction, it inhibits the rate of product formation during trypsin-catalyzed BAEE hydrolysis, resulting in a slower rate of increase in A253 per unit time (min). This reduced rate corresponds to the residual activity of trypsin after inhibition by TI (U2). Therefore, the activity of TI (TIA) can be calculated as follows: TIA = U1 - U2.
The activity of trypsin (U1) is calculated as follows:
The activity of trypsin remaining after TI inhibition (U2) is calculated as follows:
The activity of TI (TIA) is then determined as follows:
(a)Sequential Expression of Products for Delayed Toxin Release
Plasmid construction
Figure 3. Diagram of first set of verification systems circuit design
The expression of EsaⅠ (BBa_K4632026), TraR(W) (BBa_K4632028) and eGFP (BBa_K4632014) can be induced simultaneously by using arabinose.
EsaⅠ could synthesis the signaling molecule 3OC6HSL. And when the concentration of 3OC6HSL in the external environment reaches a certain threshold, EsaR will bind the 3OC6HSL to forme the EsaR-3OC6HSL complex, which binds to the promoter ptra (BBa_K2771002) to activate the expression of downstream gene -- mCherry (BBa_K4632025).
(b)Implementation of Product Expression Control
Plasmid construction
Figure 4. Diagram of second set of verification systems circuit design
The expression of LasⅠ (BBa_K4632015), LasR (BBa_K649000) and eGFP (BBa_K4632014) was induced by arabinose. LasⅠ synthesis 3-oxoC12-HSLas a signaling molecule. When the concentration of 3-oxo-C12-HSL in the external environment reaches a certain threshold, EsaⅠ will bind to the 3-oxo-C12-HSL signaling molecule to form the EsaⅠ-3-oxo-C12-HSL complex which could bind to the promoter plas (BBa_K649000) to activate the downstream expression of T4-holin (BBa_K112805) and T4 endolysin (BBa_K112806).
To verify that the pathway can operate successfully, we performed the following experiments.
The first set of validation system was transformed to Top10 strain, the arabinose was carried out as the inducer. The OD600 value, GFP and RFP fluorescence intensity were tested under microplate reader at certain time intervals. The curves were plotted with OD600 or fluorescence intensity of the GFP and RFP as the vertical axis and induced expression time as the horizontal axis. If RFP singal was detected for a period of time latter than GFP singal, the sequential expression of the product was successful.
The second set of validation system was transformed to Top10 strain as the same method described above. If the OD600 value were observed a significantly decreases after the initially increaseing, the expression of the lysis gene could be confrimed. We assessed whether the pathway could limit the maximum expression level based on the trend in eGFP signal changes. If the eGFP expression rate significantly decreases or ceases to increase after the appearance of lysis, the verification is successful. The limitation of the maximum expression level was assessed by the trend of eGFP signal change.
In parallel with our experimental work, we conducted simulation and performed genetic circuit analysis for digital logic using tools such as MATLAB and Python. The outcomes of these simulations were in accordance with our expected experimental results.
Initially, we focused on the analysis of individual bacterial cells. Our findings revealed a delayed and positively correlated expression of CPTI and Cry3A-like toxin. This delay was crucial in practice to ensure that the protease inhibitor was produced prior to the toxin, allowing for the toxin's accumulation within the red fire ant without any interference.
Subsequently, in the analysis of multiple bacterial cells, we referred to previous teams' results and developed a mathematical equation for bacterial population dynamics. By integrating this equation into our original model, we derived new insights into the total in vivo expression levels within the bacterial population. According to our findings, the bacterial population decreases rapidly following the expression of the lysis gene. However, it eventually stabilizes instead of declining to near extinction.
Furthermore, we were able to determine the peak expression levels of CPTI and Cry3A-like toxin This information served to validate whether our design adhered to the limits of the S. invicta 's survival, as well as to assess potential adverse effects on biosafety and the environment.
We utilized a third-degree function-based model for ant colony growth and simplified nursing interactions among ants. This allowed us to establish a drug enrichment model based on a system of ordinary differential equations (ODEs). factors such as the efficiency and rate of drug transmission, the number of ant species transmitting the drug, and the foraging limits of S. invicta. Additionally, we have considered the impact of the foraging upper limit on drug enrichment.
The Python's scipy package was employed to numerically solve the model we constructed. Our observations reveal a consistent increase in drug concentration within worker ants and the queen. In worker ants, drug concentration rapidly increases during the first ten days, followed by a gradual, steady rise. Notably, the rate of drug enrichment in the queen is thousands of times higher than that in worker ants. This deliberate difference ensures that worker ants are preferentially targeted by the drug before achieving the objective of selectively eliminating the queen.
Moreover, we introduced randomization into the model, simulating complex scenarios found in natural ant colonies where certain ant species exhibit significant fluctuations in their proportions. Despite these fluctuations, the drug ultimately accumulates in the direction of larvae, worker ants, and the queen. This demonstrates the robustness of our drug enrichment strategy in complex environments.
In conclusion, CPTI and Cry3A-like toxin can be secreted and expressed in a coordinated manner, both in terms of timing and spatial distribution, regulated by the quorum sensing system. This precise coordination enables the accurate targeting and elimination of the S. invicta queen.
Plasmid construction
Figure 5. Diagram of pnirB-dapA circuit design
To validate the biosafety device, the following experiments were performed:
The dapA gene was inserted behind the anaerobic promoter pnirB, ligated to pUC18T-mini-Tn7T, and then transformed to wm3064 (dapA mutant strain). The growth of △dapA/dapA- pUC18T-mini-Tn7T and △dapA was detected under aerobic and anaerobic conditions, respectively. The growth of the two strains were observed under aerobic/anaerobic conditions with or without DAP. If ∆dapA can only grow under both anaerobic or aerobic conditions with the addition of DAP; and ∆dapA/dapA- pUC18T-mini-Tn7T can grow under anaerobic conditions, then the safety device is feasible.