iGEM 2024 — Team guelph

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The Emerald Ash Borer (EAB), scientific name Agrilus planipennis, is an invasive beetle that poses a severe ecological and economic threat to ash trees in Europe and North America.

![](https://static.igem.wiki/teams/5103/emerald-ash-borer-intro-graphic.png)

**Figure 1.** Map representing Emerald Ash Borer infested areas in North America. Red pin represents Guelph, Ontario, Canada, where iGEM Guelph resides.

Since its introduction, EAB has led to devastating mortality rates, with up to 99% of ash trees in affected areas dying within a few years of infestation1. As ash trees play a crucial role in maintaining ecosystem balance by promoting soil health, influencing nutrient cycling, and providing habitats for wildlife, their loss has cascading effects on biodiversity and forest health2. Moreover, ash wood is highly valued for its strength and adaptability, making it economically important for various industries3.

Current efforts to manage EAB infestations primarily rely on chemical pesticides. However, these approaches have proven to be unsustainable in the long term. Pesticide resistance, environmental contamination, and the negative effects on non-target species and human health highlight the need for alternative solutions. The limitations of chemical pesticides inspired iGEM Guelph to explore a more sustainable and eco-friendly approach: bioinsecticides.

#### Objective of Project Ash Guard

![](https://static.igem.wiki/teams/5103/documents/engineering/ashguardlogo.png)

Our project, Ash Guard, seeks to leverage synthetic biology to protect ash trees from EAB infestations. Specifically, we aim to engineer a bioinsecticide using Saccharomyces cerevisiae (S. cerevisiae), also known as brewer's yeast, to express the Cry8Da protein, a crystal toxin derived from Bacillus thuringiensis (Bt). Cry8Da has shown specificity against insects from the Chrysomeloidea superfamily, which includes the EAB5.

#### Why Yeast?

Yeast, particularly S. cerevisiae, was chosen as the expression host for Cry8Da due to its natural symbiotic relationships with plants and insects. S. cerevisiae has been shown to produce volatile compounds that attract insects, which it uses to disperse itself into new environments6. Additionally, S. cerevisiae naturally inhabits the bark of trees, including ash trees, making it an ideal candidate for this project3. By genetically engineering S. cerevisiae to express Cry8Da, we aim to create a living barrier on ash tree bark. When EAB larvae burrow into the bark to begin their maturation process, they will encounter the Cry8Da toxin and this will lead to larval death, preventing the beetles from continuing their lifecycle and stopping the damage to ash trees before it can escalate.

![](https://static.igem.wiki/teams/5103/emerald-ash-borer-infestation-introduction.jpg)

**Figure 3.**Implementation proposal for project Ash Guard, where Cry8Da is expressed in a genetically engineered Saccharomyces cerevisiae strain. This genetically engineered strain aims to halt Emerald Ash Borer infestation.

#### Bioinsecticide as a Sustainable Solution

The use of bioinsecticides offers a sustainable alternative to conventional chemical pesticides. Bioinsecticides produced using synthetic biology, such as S. cerevisiae expressing Cry8Da, can target specific pests with minimal environmental impact. In contrast to chemical treatments that often harm non-target species and contaminate ecosystems, bioinsecticides provide a more precise pest control method. This project represents a significant step toward achieving Sustainable Development Goal (SDG) #15, which focuses on protecting the environment and promoting biodiversity.

Our approach combines advanced genetic modification techniques with a focus on ecological sustainability. The engineering success of this project will hinge on the ability to clone and express the Cry8Da protein in S. cerevisiae. Our **Engineering Success** page dives into the technical processes behind our design, including plasmid construction, protein expression, and experimental validation. Our efforts aim to provide a long-term, effective solution to EAB infestations that aligns with environmental preservation goals.

References

Herms, D. A., & McCullough, D. G. (2014). Emerald ash borer invasion of North America: History, biology, ecology, impacts, and management. Annual Review of Entomology, 59, 13-30.

Mortensen, L. H., Cruz-Paredes, C., & Vestergård, M. (2019). Ash application enhances decomposition of recalcitrant organic matter. Soil Biology & Biochemistry, 135, 316-322.

Arbab, N., Grabosky, J. R., Leopold, R. (2022). Economic assessment of urban ash tree management options in New Jersey. Sustainability, 14(4), 2172.

McKinney, L.V., Nielsen, L. R., Collinge, D. B., Thomsen, I. M., Hansen, J. K., & Kjær, E. D. (2014). The ash dieback crisis: Genetic variation in resistance can prove a long‐term solution. Plant Pathology, 63(3), 485-499.

Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Bravo, A., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642

Christiaens, J. F., Franco, L. M., Cools, T. L., De Meester, L., Michiels, J., Wenseleers, T., Hassan, B. A., Yaksi, E., & Verstrepen, K. J. (2014). The fungal aroma gene ATF1 promotes dispersal of yeast cells through insect vectors. Ecology and Evolution, 4(19), 3905-3919.

Snapp, E. L. (2009). Fluorescent Proteins: A Cell Biologist’s User Guide. Trends in Cell Biology, 19(11): 649-655.

United Nations. (2015). Transforming our world: The 2030 Agenda for Sustainable Development.

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# 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.

![](https://static.igem.wiki/teams/5103/documents/engineering/ashguardvisualization1.png)

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

## Design:

---

![](https://static.igem.wiki/teams/5103/documents/engineering/modelingproteininteraction.jpg)

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
death4,5.

### 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
* pET28a for E. coli
* pYES2 for S. cerevisiae

#### pGC004 for E. coli

![](https://static.igem.wiki/teams/5103/documents/engineering/pcgcryplasmid.png)

**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:

![](https://static.igem.wiki/teams/5103/documents/engineering/pet28acryplasmid.png)

**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

![](https://static.igem.wiki/teams/5103/documents/engineering/pyes2-cryplasmid.png)

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D).

* pGC004 for E. coli
* pET28a for E. coli
* pYES2 for S. cerevisiae

#### pGC004 for E. coli

![](https://static.igem.wiki/teams/5103/documents/results/cry8da-in-e-coli-protein-1.jpg)

**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.

#### 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

![](https://static.igem.wiki/teams/5103/documents/engineering/cry8dainyeast.jpg)

**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.

![](https://static.igem.wiki/teams/5103/documents/engineering/ashguardimplementation.jpg)

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:

1. 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
2. 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

![](https://static.igem.wiki/teams/5103/documents/engineering/ashguardvisualization3.png)

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:

1. E. coli PCG004 (empty vector)
2. E. coli PCG-CRY
3. S. cerevisiae PYES2 (empty vector)
4. S. cerevisiae PYES2-CRY
5. 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

![](https://static.igem.wiki/teams/5103/documents/engineering/sdspageanalysis.png)

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

---

![](https://static.igem.wiki/teams/5103/documents/engineering/ashguardlogo.png)

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.

References

1. Koski, T .M., Zhang, B., Mogouong, J., Wang, H., Chen, B., Li, H., Bushley, K. E., & Sun, J. (2024). Distinct metabolites affect the phloem fungal communities in ash trees (Fraxinus spp.) native and nonnative to the highly invasive emerald ash borer. Plant, Cell & Environment. https://onlinelibrary.wiley.com/doi/abs/10.1111/pce.14996

2. Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Bravo, A., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642. https://doi.org/10.3390/toxins12100642

3. Bravo, A., Gill, S. S., & Soberón, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423-435. https://doi.org/10.1016/j.toxicon.2006.11.022

4. Yamaguchi, T., Bando, H., & Asano, S. (2013). Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica. Journal of Invertebrate Pathology, 113(2), 123-128. https://doi.org/10.1016/j.jip.2013.03.006

5. Yamaguchi, T., Sahara, K., Bando, H., & Asano, S. (2010). Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. Journal of Invertebrate Pathology, 105(3), 243-247. https://doi.org/10.1016/j.jip.2010.07.002

6. Held, B. W., Simeto, S., Rajtar, N. N., Cotton, A. J., Showalter, D. N., Bushley, K. E. & Blanchette, R. A. (2021). Fungi associated with galleries of the emerald ash borer. Fungal Biology, 125(7), 551-559. https://doi.org/10.1016/j.funbio.2021.02.004

7. Studier, F. W., & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology, 189(1), 113-130. https://doi.org/10.1016/0022-2836(86)90385-2

8. Urh, M., Simpson, D., & Zhao, K. (2009). Chapter 26 Affinity Chromatography: General Methods. Methods in Enzymology, 463, 417-438. https://doi.org/10.1016/S0076-6879(09)63026-3

9. Lallemand Inc. (2024). TreeAzin Systemic Insecticide – BioForest. Bioforest.ca. https://bioforest.ca/en/canada/product-details/treeazin-systemic-insecticide/

10. Fatima, K., Naqvi, F. & Younas, H. (2021). A Review: Molecular Chaperone-mediated Folding, Unfolding and Disaggregation of Expressed Recombinant Proteins. Cell Biochemistry and Biophysics 79, 153-174. https://doi.org/10.1007/s12013-021-00970-5

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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.

![](https://static.igem.wiki/teams/5103/purification-of-cry8da-future-directions.png)

**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.

![](https://static.igem.wiki/teams/5103/igem-swiss-model-screenshot.png)

**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

![](https://static.igem.wiki/teams/5103/extraction-of-cry8da-from-yeast-simplified-workflow.png)

**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.

![](https://static.igem.wiki/teams/5103/antibiotic-selection-marker-vs-auxotrophic-selection-marker.png)

**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.

References

Raymond, B., Johnston, P. R., Wright, D. J., Ellis, R. J., Crickmore, N., & Bonsall, M. B. (2010). A midgut microbiota is not required for the pathogenicity of Bacillus thuringiensis to diamondback moth larvae. Environmental Microbiology, 12(10), 2931-2938.

Duan, J. J., Bauer, L. S., Van Driesche, R. G., & Gould, J. R. (2018). Progress and challenges of protecting North American ash trees from the emerald ash borer using biological control. Forests, 9(3), 142.
Griffiths, B. S., Kuan, H. L., Ritz, K., Glover, L. A., McCaig, A. E., & Fenwick, C. (2007).

Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642.

Wang, W., Vinocur, B., Shoseyov, O., & Altman, A. (2014). Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science, 9(5), 244-252.

Wright, G. D. (2010). Antibiotic resistance in the environment: A link to the clinic? Current Opinion in Microbiology, 13(5), 589-594.

Zou, M., Xin, B., Sun, X., Lin, R., Lu, J., Qi, J., Xie, B., & Cheng, X. (2023). URA3 as a Selectable Marker for Disruption and Functional Assessment of PacC Gene in the Entomopathogenic Fungus Isaria javanica. Journal of Fungi, 9(1), 92.

Snapp, E. L. (2009). Fluorescent Proteins: A Cell Biologist’s User Guide. Trends in Cell Biology, 19(11): 649-655.

Health Canada. (2008, June 5). Health Canada's Role in the Regulation of Products from Biotechnology. Canada.ca.

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# 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.

![Streak plate of PCG-Cry8Da](https://static.igem.wiki/teams/5103/documents/results/cry8da-in-e-coli-protein-1.jpg)

**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.

![0.8% Agarose gel electrophoresis of PCG-CRY plasmid](https://static.igem.wiki/teams/5103/documents/results/results-figure-1.jpg)

**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.

![0.8% Agarose gel electrophoresis of PRS-316](https://static.igem.wiki/teams/5103/documents/results/results-figure-2.jpg)

**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

![Transformant pYES2-Cry8Da in S. cerevisiae BY4742](https://static.igem.wiki/teams/5103/documents/results/cry8da-in-yeast.jpg)

**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](https://static.igem.wiki/teams/5103/documents/results/dead-beetles-results-page.jpg)

**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.

![10% SDS-PAGE analysis of Cry8Da expression in E. coli](https://static.igem.wiki/teams/5103/documents/results/sds-page.jpg)

**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.

References

1. Zou, M., Xin, B., Sun, X., Lin, R., Lu, J., Qi, J., Xie, B., & Cheng, X. (2023). URA3 as a Selectable Marker for Disruption and Functional Assessment of PacC Gene in the Entomopathogenic Fungus Isaria javanica. Journal of Fungi, 9(1), 92. https://doi.org/10.3390/jof9010092

2. Yamaguchi, T., Sahara, K., Bando, H., & Asano, S. (2010). Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. Journal of Invertebrate Pathology, 105(3), 243-247. https://doi.org/10.1016/j.jip.2010.07.002

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