The next steps for Ash Guard include proving the toxicity of the Cry8Da protein, refining the expression system, and ensuring safe, real-world application. These future steps are crucial to bridge the gap between Ash Guard's development in the lab and its potential for practical use in protecting ash trees from emerald ash borer (EAB) infestations.
Purification and testing of the Cry8Da protein:
To fully assess the effectiveness of Cry8Da, the next step involves purification of the protein from recombinant Escherichia coli (E. coli) DH5α. This approach, rather than introducing recombinant bacteria directly into the environment, would allow for in vitro testing of purified Cry8Da protein. In vitro tests are essential because they eliminate potential interactions between the bacterial host and the environment, providing a clearer picture of Cry8Da's toxic effects.
Figure 1.Simplified diagram of purification process. Escherichia coli cells will need to be lysed, centrifuged to separate the supernatant (proteins) from cell debris and then the His-tag will be used to bind to a matrix column, then eluted.
Once purified, the Cry8Da protein can be tested on adult EABs, larvae, and non-target insects using controlled toxicity assays. These assays would involve varying Cry8Da concentrations and recording mortality rates in both EAB and non-target species. Bioinformatics tools, such as BLAST and Swiss-Model, can predict possible off-target effects on insects closely related to EAB.2 BLAST allows us to compare the Cry8Da sequence to those of other insects, identifying any similarities hat might indicate potential off-target effects on non-target species. Swiss-Model can be used to model the 3D structure of Cry8Da, helping predict how it interacts with the receptors in EAB gut cells and related species. These predictions are critical, as they inform regulatory decisions and help assess the protein’s broader ecological impact.
Figure 2. Swiss-model website showing some of its protein modeling options.
Understanding the dosage requirements and potential sex-specific differences in toxicity is vital for optimizing future treatments. In addition, comprehensive toxicity data is required to seek regulatory approval from agencies like the Canadian government before field trials or real-world applications can begin3. This step is key to transitioning Ash Guard from lab development to an environmentally safe solution and is discussed in more detail on the human practices page.
Demonstrating protein expression
To confirm the successful expression of Cry8Da in E. coli, SDS-PAGE analysis is required. The SDS-PAGE will display the molecular weight of the expressed Cry8Da protein, which is expected to be approximately 130 kDa.4 This will serve as evidence that the Cry8Da protein has been expressed in the host organism.
Control:
A control group with E. coli transformed with an empty plasmid (pGC004 without Cry8Da) will be run alongside to demonstrate that any observed band at 130 kDa is due to Cry8Da 4 and not other proteins present in the cell.
Outcome:
The appearance of a distinct band at 130 kDa on the SDS-PAGE gel would confirm successful Cry8Da expression.
Due to time constraints, we were unable to extract the protein from S. cerevisiae for SDS-PAGE analysis. In future work, efforts should focus on identifying or developing a protocol for the efficient extraction of Cry8Da from S. cerevisiae. Cry proteins similar to Cry8Da have been extracted from yeast before, providing a starting point for designing an extraction protocol for this project
Figure 3. Simplified workflow of Cry8Da protein extraction from Saccharomyces cerevisiae.
Assessment of expression system and optimization of plasmid design:
Future work should also involve exploring alternative expression systems for Cry8Da that optimize safety and functionality. One promising direction is to develop inducible expression systems, where Cry8Da is only produced in the presence of EAB-related signals. This would minimize any unintended effects on non-target organisms and ensure Cry8Da is expressed only when necessary. For example, an inducible promoter that responds to plant stress signals or insect-related pheromones could be designed.
Another potential system involves plasmids with alternative markers to antibiotic resistance genes. While antibiotic resistance markers are commonly used in labs for selection, their use in the environment poses significant risks due to the ongoing global health challenge of increasing antibiotic resistance6. The accidental release of a genetically modified organism (GMO) containing an antibiotic resistance marker could exacerbate this problem, making it essential to explore alternatives.Another potential system involves plasmids with alternative markers to antibiotic resistance genes. While antibiotic resistance markers are commonly used in labs for selection, their use in the environment poses significant risks due to the ongoing global health challenge of increasing antibiotic resistance6. The accidental release of a genetically modified organism (GMO) containing an antibiotic resistance marker could exacerbate this problem, making it essential to explore alternatives.
Figure 4. SPlasmid maps identifying Antibiotic markers (Ampicillin) versus Auxotrophic markers (URA3). Auxotrophic markers can be used instead of antibiotic markers to prevent contributing to the growing antibiotic resistance crisis.
Possible alternative markers for use in environmental applications include:
- Auxotrophic markers: These markers can complement specific nutrient deficiencies in the host organism. For instance, the URA3 marker used in yeast is an auxotrophic marker that allows selection in uracil-deficient media without the risk of antibiotic resistance transfer.
- Fluorescent markers: Another option could be the use of a safe fluorescent protein marker for selection and visualization without relying on antibiotics.
Plasmid Design Optimization:
also designed a plasmid system using the pET28a plasmid, which includes a 6x histidine tag for protein purification. While this worked in silico, we were unsuccessful in cloning the Cry8Da gene into pET28a in the lab. Moving forward, it would be beneficial to optimize this plasmid to facilitate Cry8Da purification and further testing. This plasmid could be tested alongside the pYES2 system for S. cerevisiae and pGC004 for E. coli.
Environmental Safety and Real-World Application
Transitioning Ash Guard into a real-world solution requires more than just lab success. Future studies should focus on:
- Evaluating the environmental safety of Cry8Da by conducting detailed ecological assessments of non-target effects and confirming its specificity to EAB.
- Regulatory compliance: Comprehensive environmental and toxicity studies will be required to meet the standards set by government regulatory bodies, such as the Canadian Food Inspection Agency and Health Canada, for the release of any bioengineered organism or product.
Conclusion
The future of Ash Guard lies in advancing beyond the lab and ensuring its safe, effective application in real-world environments. The next steps include purifying the Cry8Da protein for more detailed toxicity testing, optimizing the plasmid design for safe and controlled expression, and demonstrating environmental safety. By pursuing these directions, Ash Guard can be used to offer an innovative, sustainable solution to combat EAB infestations and protect ash trees from further destruction.
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