Project Ash Guard

The purpose of our project is to engineer Saccharomyces cerevisiae (S. cerevisiae) BY4742 to express the Cry8Da protein, which will be tested for its insecticidal effects against the Emerald Ash Borer (EAB). The EAB is a highly destructive pest responsible for devastating ash tree populations. By utilising S. cerevisiae, a yeast species that naturally lives on the bark of ash trees¹, we aim to develop a biological defence mechanism against EAB infestations.

Figure 1. Workflow of wet lab component of project Ash Guard documenting experimental design to protein expression.

Design:

This project involved engineering Escherichia coli (E. coli) DH5α and S. cerevisiae BY4742 to express the Cry8Da protein as a novel bioinsecticide. Research has shown that Cry8Da effectively kills insects in the Coleoptera order.² The protein works by binding to receptors in the insect's midgut, leading to pore formation and eventual cell death.^2,3 The toxic domain of the Cry8Da protein is thought to be its 54 kDa active fragment, which is generated through proteolytic cleavage.^4,5

Figure 2. Model of Cry8Da protein in a coiled structural motif using AlphaFold. Cry8Da is coloured using blue and green. The green part corresponds to the 54kDa portion of the protein. This part of the protein interacts the most with ß-glucosidase (pink).

Why Cry8Da?

Cry8Da is part of the Cry protein family produced by Bacillus thuringiensis (Bt), which is well-known for targeting insects³. It has shown specific toxic activity against beetle larvae, including members of the Chrysomeloidea superfamily, which the EAB belongs to². Using S. cerevisiae as the chassis allows for a sustainable, environmentally integrated solution by creating a living, self-replicating bioinsecticide that exists naturally on ash trees.⁶ Cry8Da is activated through proteolytic cleavage, and the resulting fragments are essential for the toxin’s insecticidal action, with different domains responsible for binding to receptors (C-terminal) and pore formation (N-terminal) in the gut cell of the beetle⁵. Cry8Da gets cleaved into 3 fragments: 64kDa, 54kDa and 8kDa. The 54kDa and the 8kDa fragments are generated by intramolecular cleavage at the loop between the α3 and α4 helices of domain I. The 54kDa fragment is the primary binding fragment responsible for interacting with receptors in the insect's gut such as ß-glucosidase. This interaction is what causes the mechanism of action leading to pore formation in the cell membrane and insect death^4,5.

Plasmid Design and DNA Construct Assembly

We used multiple plasmids in this project, each serving a distinct purpose. Below is a breakdown of what we aimed to achieve with each plasmid and the challenges we encountered.

pGC004 for E. coli

Figure 3. PCG-CRY plasmid components were developed using SnapGene.

The pGC004 plasmid was used to express Cry8Da in E. coli. This plasmid was selected because it is a shuttle vector capable of replicating in both E. coli and B. subtilis. Also, this plasmid allows us to express our protein E. coli DH5α because its promoter does not require the T7 RNA polymerase, which is absent in DH5α cells. Instead, PCG004 uses a promoter that can be recognized by the native RNA polymerase of E. coli, enabling gene expression without the need for additional machinery.⁷ Cry8Da was inserted into the plasmid, and the expression was controlled using a Lac operon. In addition to cloning Cry8Da into PCG004 we aimed to use a mScarlet Fluorescent tag downstream of Cry8Da to measure gene transcription levels. While we were successful in combining the Cry8Da and mScarlet, we faced challenges with the cloning of a combined Cry8Da-mScarlet fragment in this plasmid.

Plasmid Components: BBa_K5103001

Cry8Da gene: Insecticidal protein gene that has been modified to be transformed into PCG004. It corresponds to Part BBa_K5103000 in the Parts registry.
Ampicillin resistance: Selection marker for transformed E. coli.
Lac operon: IPTG-inducible promoter, controls Cry8Da expression in E. coli.

pET28a for E. coli:

Figure 4. pET28a-CRY plasmid components developed in SnapGene.

We hoped to use the pET28a plasmid due to its 6x histidine tag. The addition of this tag would allow us to easily purify the Cry8Da protein from E. coli using affinity chromatography,⁸ which simplifies the downstream protein purification process. Affinity chromatography works by exploiting the specific interaction between the histidine tag and a metal ion, like nickel, bound to a resin. The tagged protein binds to the metal, while other proteins are washed away, allowing for selective purification of the target protein.⁸

Plasmid Components:

Cry8Da gene: Insecticidal protein gene that corresponds to Part BBa_K5103000 in the registry.
6x histidine tag: For protein purification.
Kanamycin resistance: Selection marker for E. coli.
IPTG-inducible promoter: Controls Cry8Da expression in E. coli.

pYES2 for S. cerevisiae

Figure 5. PYES2-CRY plasmid components developed in SnapGene.

The pYES2 plasmid was used to express Cry8Da in S. cerevisiae. This plasmid contains a galactose-inducible GAL1 promoter, allowing us to control protein expression in S. cerevisiae. The ultimate goal is to use S. cerevisiae as a natural, sustainable defence mechanism against EAB due to its ability to inhabit the bark of ash trees.

Plasmid Components: BBa_K5103004

Cry8Da gene: Insecticidal protein gene. Primers were used to introduce PYES2-specific cut sites. Part BBa_K5103003 in the registry.
URA3 selection marker: Allows S. cerevisiae growth on media lacking uracil.
GAL1 promoter: Induces Cry8Da expression in S. cerevisiae in the presence of galactose.

In Silico Verification:

Before proceeding with wet lab experiments, we performed in silico verification using SnapGene to confirm the integration of the Cry8Da gene into each plasmid. This involved:

Selecting appropriate restriction enzyme (BsaI, HindIII, XhoI and NdeI) cut sites for seamless cloning.
Designing primers for successful amplification using Snapgene. See Parts: BBa_K5103006 and BBa_K5103007.
Simulating the ligation process to ensure proper orientation and sequence integration.

All plasmid designs were successful in silico, but challenges arose during wet lab implementation, as detailed below.

Build:

Our plasmid expression systems were designed to express Cry8Da in E. coli and S. cerevisiae. The plasmids were carefully constructed to include selection markers (ampicillin and kanamycin for E. coli, URA3 for S. cerevisiae) and inducible promoters (IPTG for E. coli, GAL1 for S. cerevisiae). This section will summarise the challenges and successes of our project’s experimentation. For a more in-depth methodology, explore our lab notebook and protocols on the Contribution page.

pGC004 for E. coli

Figure 6. Streak plate of PCG-Cry8Da transformant colonies grown on ampicillin selective media.

Challenges:

Although we successfully validated the Cry8Da-mScarlet construct in silico, the cloning process into pGC004 was unsuccessful. Due to time constraints, we decided to proceed without the mScarlet tag. However, we were able to successfully clone Cry8Da alone into pGC004, which was then used for further testing.

In Silico Success:

In silico analysis confirmed successful integration of the cry8Da-mScarlet fragment into pGC004, but the failure in wet lab cloning forced us to abandon this approach.

pET28a for E. coli

Challenges:

Although in silico verification confirmed the successful integration of the Cry8Da gene into pET28a, wet lab cloning attempts were unsuccessful. This hindered our ability to extract and purify the protein using the 6x histidine tag.

In Silico Success:

Similar to pGC004, the integration of the Cry8Da gene into pET28a was successful in computational simulations but failed during wet lab experiments.

pYES2 for S. cerevisiae

Figure 7. Transformant pYES2-Cry8Da in S. cerevisiae BY4742. Colonies were
selected for using URA3 and uracil-deficient, selective media

Success:

The plasmid was successfully transformed into S. cerevisiae, and we were able to confirm this ligation using agarose gel electrophoresis. Additionally, yeast cells successfully grew in galactose induction medium. However, further testing with Sodium Dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) will need to be done to confirm protein production levels.

The ultimate goal was to use S. cerevisiae as a natural insecticide integrated into the ecosystem of ash trees. The presence of S. cerevisiae on the bark, coupled with the expression of Cry8Da, would form a living barrier that could protect ash trees from EAB infestations without the need for repeated chemical treatments.

Figure 8. Proposal for Ash Guard implementation. There are two possible implementations of Ash Guard - Saccharomyces cerevisiae that would express Cry8Da and exist on tree bark, and Escherichia coli that would be used to biomanufacture Cry8Da and that the extracted protein would be made into a spray that would be sprayed on the tree.

Test:

We will test the system’s ability to express the Cry8Da protein by:

Running SDS-PAGE to confirm the presence of the Cry8Da protein in E. coli.
- We induced the expression of Cry8Da with IPTG at OD600=~0.800. We incubated two tubes (one with IPTG and one without) in a 15°C shaking incubator overnight and 2 tubes (one with IPTG and one without) in a 37°C shaking incubator overnight.
- The following day the tubes were collected and proteins from the samples were extracted. Each sample provided us with a soluble and insoluble protein sample.
- Protein extraction and SDS-PAGE was not repeated for S. cerevisiae, due to time constraints and lack of certain reagents required for the procedure
Feed our successfully cloned organisms to adult EAB
- Samples will be diluted to an OD600 = 1.0 and each test group will receive ash leaves sprayed three times with the appropriate sample.
- Behaviour of beetles will be observed and toxicity will be measured in the time that it takes for the beetles to decease determined by no further movement.
- Mortality within test groups will be compared to the controls and the other test groups.

Experimental Design:

During optical density (OD) measurements, SDS-PAGE and toxicity testing of Ash Guard on adult EABs.

SDS-Page Attempt 1

Our first attempt at running an SDS-PAGE was unsuccessful and no band at 130kDa was observed.⁵ This could be due to potential error during the protocol for inducing the expression of the protein as it was our first attempt, or simply due to low expression levels that were not sufficient to be observed on the gel.

Toxicity Testing Attempt 1

Ash tree leaves were collected from the University of Guelph campus, washed with water, and surface dried. We ordered 15 female and 15 male adult emerald ash borers (EAB), which were separated into 10 groups, each consisting of 3 beetles.

Figure 9. Experimental design and set-up of introducing Cry8Da protein to Emerald Ash Borers (EABs).

Experimental groups:

E. coli PCG004 (empty vector)
E. coli PCG-CRY
S. cerevisiae PYES2 (empty vector)
S. cerevisiae PYES2-CRY
Water control (negative control)

Each experimental group consisted of 3 male and 3 female adult EAB. The beetles were kept at 4°C prior to testing to keep them in a dormant state. In the biosafety cabinet (BSC), two similar-sized leaves were sprayed three times with the appropriate sample (refer to experimental groups above). Immediately after spraying, the corresponding beetle containers were taken out of the 4°C incubator, and the leaves were placed into the EAB containers to avoid the possibility of the beetles waking up too early, risking escape, before the containers were sealed. Several steps were taken to ensure consistency and accuracy throughout the process:

All beetle containers and equipment were pre-labeled to minimise mistakes and ensure a faster transition, reducing the time difference between the first and last leaves being placed in the containers.
Between each experimental group, the BSC, tweezers, and hands were thoroughly sprayed with 70% ethanol to prevent cross-contamination between the experimental groups.
Special care was taken to ensure that the spray bottles dispensed the same volume of sample for each leaf. To achieve this, each spray bottle was tested by spraying into a waste beaker before use, ensuring that the spray tube was clear and functioning properly.

Challenges in Toxicity Testing

One major challenge in testing was the premature death of the EABs during our initial tests, as they were fed pesticide-treated leaves. In Guelph, trees are treated with TreeAzin, an injectable systemic pesticide.⁹ Due to the systemic nature of TreeAzin, despite it being injected in the trunk of trees, it will be present in the leaves of treated ash trees and was a confounding variable and thus we cannot confidently determine whether the death of the EABs was due to our bioinsecticide effectively expressing the Cry8Da protein or not.

SDS-PAGE Attempt 2

Attempts to visualise the expression of Cry8Da in E. coli were repeated however, once again no bands were observed at 130kDa where Cry8Da should be present.5 This could be due to low expression of Cry8Da. Further testing is required to confirm this, such as switching promoters, plasmid backbone and codon optimization. Future iterations of testing are discussed below.

Figure 10. 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 (Precision Plus Protein™ All Blue Prestained Protein Standard). Cry8Da (~130 kDa) was expected in Lanes 1, 1S, 3, and 3S. No clear band corresponding to Cry8Da was observed at 130 kDa.

Learn

Our project follows the Design-Build-Test-Learn cycle, an important approach that allows us to improve on each phase of the project based on data and feedback gathered.

SDS-PAGES for Protein Expression

One important step in determining the success of Cry8Da expression in E. coli and S. cerevisiae was performing SDS-PAGE analysis. Unfortunately, as both of our SDS-PAGE attempts were unsuccessful we were unable to confidently determine the protein expression of Cry8Da within project Ash Guard. The possible reasons for the failed SDS-PAGEs include:

Insufficient protein expression: The failed attempt to detect Cry8Da through SDS-PAGE may have been due to low levels of expression in E. coli this could be solved by optimising the promoter strength or using a different method of detection such as western blotting, to detect low levels of protein. This will however require us to tag the protein using pET28a, which was a failed attempt.
Protein misfolding: Another hypothesis is that Cry8Da may not have properly folded in the host organism, resulting in an inactive or non-functional protein. To fix this we could attempt to express molecular chaperons that assist in protein folding or modify the expression conditions.¹⁰

Failures and lessons learned:

Although many of our experiments did not proceed as expected, these failures provided valuable insights for future attempts. For example, cloning failures in earlier stages of the project allowed us to change certain methods or angles we were using. One commonly occurring change was simple adjustments of reagents, incubation times and temperatures. These adjustments helped us have better success with digesting our fragments to then ligate and transform them. If we had the opportunity to repeat the summer experiments, we would focus on:

Optimising cloning protocols earlier in the process to avoid setbacks
Testing a different approach to measuring gene activity by using a different fluorescent marker, which may offer better stability and expression in our host organism.
Exploring different strategies to optimise the system such as promoter strength (using a stronger inducible promoter, or codon optimization to increase the efficiency of transcription in both E. coli and S. cerevisiae), constitutive expression (the current expression system in S. cerevisiae relies on the GAL1 promoter, which would not be practical in real-world application, so the use of constitutive promoters 10 or promoters that environmental or molecular signals could activate should be looked into.

As stated earlier we were unable to determine whether project Ash Guard was effectively expressing Cry8Da. This failure in the wet lab led to several important considerations for future tests:

Pesticide-free leaves: our initial testing was negatively impacted by the fact that the beetles were exposed to leaves previously treated with pesticides. Future experiments will use pesticide-free leaves to ensure that any mortality observed in the EAB beetles is directly attributable to Cry8Da, rather than pesticide residues.
Protein expression and folding: another aspect we will need to investigate further is the expression of the Cry8Da proteins and whether the Cry8Da protein was properly folded and functional after expression. Incorrect folding could impair the insecticidal activity of Cry8Da.

After reconducting our tests, we will compare our data with the design specifications. If expression is lower than expected, we will consider modifying the system, such as adjusting promoter strength or codon optimization. Another consideration is that due to all these failed testing attempts there was limited time to conduct further testing of our final product - Ash Guard. Should we be able to reattempt this project, we would also conduct tests to model the behaviour of the genetically modified S. cerevisiae strain expressing Cry8Da (Ash Guard) compared to the non-genetically modified S. cerevisiae strain.This testing will explore how each strain interacts with the environment, in terms of growth rates, and overall impact on the ecosystem. The generated data would be particularly important for assessing the environmental safety of the final product and ensuring that the Cry8Da expressing S. cerevisiae will not negatively impact non-target organisms or the ecosystem. It would also show if there was a difference in expression between S. cerevisiae and E. coli.

This data could be supported by using biocontainment strategies to ensure the S. cerevisiae expressing Cry8Da only survives in the specific conditions it is intended to, limiting its spread, however there may be a lot of challenges associated with this, which would be worth the research. As we are proposing the use of a genetically modified organism (GMO) in real-world settings, is it imperative to ensure that the expression of Cry8Da is safe/minimal for non-target organisms, to avoid unintended environmental impacts.

Our entire dataset and methodology will be documented and made available through our iGEM team's wiki to ensure transparency and reproducibility.

Conclusion

The Design-Build-Test-Learn cycle underpins our approach to engineering Cry8Da expression in E. coli and S. cerevisiae. Despite some setbacks in cloning and protein extraction, we are moving forward with testing the Cry8Da protein's efficacy against the EAB. Our goal is to use S. cerevisiae as a natural biocontrol mechanism on ash trees, providing a sustainable, long-term solution to EAB infestations.

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