Results and Discussion

Throughout our work on Project Ash Guard, we encountered a variety of challenges related to plasmid compatibility, gene expression, and the overall success of transforming Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) with the Cry8Da gene. These results, while at times disappointing, provide valuable insight into the obstacles of engineering biological systems and offer a roadmap for future research.

Successful Cloning and Transformation

One of the key successes of the project was the successful cloning of the Cry8Da gene into two different plasmid systems: pGC004 for E. coli and pYES2 for S. cerevisiae. After cloning, we successfully transformed these plasmids into their respective organisms, achieving our goal of cloning Cry8Da in both bacterial and yeast expression systems. Thus, bringing us one step closer to creating an expression system producing a Cry8Da bioinsecticide.

pGC004 for E. coli

Cry8Da was successfully cloned into the pGC004 plasmid and subsequently transformed into E. coli DH5α cells.
The transformation was verified, and E. coli colonies were grown on ampicillin selective media, confirming the presence of the PCG-Cry8Da plasmid.

Figure 1. Streak plate of PCG-Cry8Da transformant colonies grown on ampicillin selective media.

Additionally, the transformed colonies were miniprepped and digested with the same enzymes. An agarose gel electrophoresis was performed to confirm the successful ligation of Cry8Da in PCG004.

Figure 2. 0.8% Agarose gel electrophoresis of PCG-CRY plasmid digested with BsaI compared to the undigested PCG-CRY plasmid. Digestion shows successful ligation of the Cry8Da gene in pCG004 plasmid backbone. Results showed a band at ~8000bp corresponding with PCG004 and ~3400 bp corresponding with Cry8Da.

pYES2 for S. cerevisiae

Similarly, Cry8Da was cloned into the pYES2 plasmid and successfully transformed into S. cerevisiae BY4742.
pYES2-CRY plasmid was first transformed into E. coli, and was selected with an ampicillin-resistant marker.
The successfully transformed colonies were then miniprepped, digested and run on an agarose gel to confirm the ligation of Cry8Da in pYES2.

Figure 3. 0.8% Agarose gel electrophoresis of PRS-316 plasmid with Cry8Da and PYES2-Cry8Da digested with HindIII and XhoI. Ligation of Cry8Da in PRS-316 was unsuccessful as only bands observed were at ~5000 bp. Ligation of Cry8Da in pYES2 was successful as shown above. Faint lines at ~3400 bp correspond with Cry8Da and ~5800 bp with pYES2.

S. cerevisiae colonies were selected using URA3 as the selection marker by growing on uracil deficient media, verifying the successful transformation of the cells with pYES2-CRY. The URA3 gene encodes for orotidine-5 phosphate decarboxylase (OMP decarboxylase) and helps in the uracil pathway so without this gene the S. cerevisiae cannot synthesize uracil and needs supplemented uracil or uridine.¹

Figure 4. Transformant pYES2-Cry8Da in S. cerevisiae BY4742. Colonies were selected for using URA3 and uracil-deficient, selective media.

Testing on Emerald Ash Borers

After successfully transforming the Cry8Da gene into E. coli and S. cerevisiae, we proceeded with testing on EAB adult beetles to evaluate the toxicity of Cry8Da. Despite our successful transformations, these tests did not yield conclusive results. The EABs were fed leaves from ash trees that had been treated with pesticides, which led to their premature death. This complication meant we could not assess the effectiveness of Cry8Da in killing the beetles, as the results were confounded by the pesticide exposure.

Postmortem photograph of Emerald Ash Borers (EAB) used during project Ash Guard’s toxicity

Figure 5. Transformant pYES2-Cry8Da in S. cerevisiae BY4742. Colonies were selected for using URA3 and uracil-deficient, selective media.

Future Plans:

We plan to repeat the beetle testing, ensuring that pesticide-free leaves are used, allowing us to isolate the effects of the Cry8Da protein.
Additional controlled testing on EAB larvae and non-target species will also be conducted to evaluate the protein's specificity and efficacy fully. More details can be found on the Future Directions page.

Cry8Da Expression Difficulties and Future Directions

Although we successfully transformed the Cry8Da gene into E. coli and S. cerevisiae, we have yet to confirm the protein expression. Due to time constraints, we were unable to perform SDS-PAGE analysis, which would have allowed us to verify Cry8Da production and assess its expression level.

SDS-PAGE for Protein Expression in E. coli

We plan to conduct an SDS-PAGE analysis using the pGC004-Cry8Da transformed E. coli cells.
This test will:

Confirm the presence of the Cry8Da protein by identifying a band at the expected molecular weight of 130 kDa.²
Use control samples, including E. coli transformed with the empty pGC004 plasmid, to ensure that any observed band corresponds specifically to Cry8Da.

Conducting this analysis is essential for verifying protein production and ensuring that the Cry8Da protein is being expressed as expected in the bacterial host.

Figure 6. 10% SDS-PAGE analysis of Cry8Da expression in E. coli. Samples include soluble (S) and insoluble fractions from cultures grown overnight at 15°C (Lanes 1S, 1, 2S, 2) and 37°C (Lanes 3S, 3, 4S, 4) with IPTG induction. Lane L contains the molecular weight marker. Cry8Da (~130 kDa) was expected in Lanes 1, 1S, 3, and 3S. No clear band corresponding to Cry8Da was observed at 130 kDa.

While we achieved significant progress with the cloning and transformation of Cry8Da, other parts of the project did not proceed as smoothly:

mScarlet Fluorescent Tag: Attempts to combine Cry8Da with the mScarlet fluorescent protein for real-time activity tracking were unsuccessful. We were able to combine Cry8Da, with mScarlet in PIDT (IDT plasmid backbone), however colonies failed to survive after ligating Cry8Da-mScarlet with PCG004 and transformation in E. coli DH5α. This combination worked in silico, but failed during wet lab cloning in E. coli. Time constraints prevented further troubleshooting of this approach.
pET28a for Protein Purification: The pET28a plasmid includes a 6x histidine tag, which was intended to allow for the purification of Cry8Da, however, despite successful in silico validation, we were unable to successfully clone Cry8Da into this plasmid in the wet lab. This setback meant that protein purification could not be achieved through the pET28a system.

Conclusion

Although these experiments faced challenges and some even failed, the insights gained from these experiments are valuable for future research. Understanding the incompatibility between Cry8Da and certain plasmid backbones will help researchers avoid similar pitfalls, and our successful transformations offer a foundation for future experimentation.

In summary, while not all experiments were successful, the cloning and transformation of Cry8Da into E. coli and S. cerevisiae represent significant milestones for project Ash Guard. These findings will help shape future bioinsecticide research and development, guiding others in their efforts to combat EAB infestations with environmentally sustainable solutions.