Proof of Concept

Following our work for Engineering Success and Proposed Implementation, we did further tests to confirm our project functioned as it was meant to. This included tests of toxicity, and to ensure we had not lost key functionality of our bacterial chassis.

Proof of Concept

As previously described in our Engineering Success Page, Project Ceres is meant to create a novel biopesticide for use in greenhouses through efficiently and specifically expressing the cytotoxic proteins Cyt1Aa and Cyt2Ba in Bacillus subtilis. For experimental success, our wet lab goals are split up between Engineering Success and Proof of Concept. In short our specific outcomes were:

Summarizing Engineering Success, our team was able to achieve all 5 goals and ultimately created B. subtilis strains that were able to have increased insecticidal activity against D. melanogoster when compared to non-targeting controls. These strains were also able to fluoresce the expected colours as observed by the results of our fluorescence microscopy (Table 1).

With the strains initially validated to be expressing the constructs, in our Proof of Concept section we intend to describe our further profiling of the strains. Our experiments are divided into two broad categories: in vitro and in vivo assays. Our first three in vitro assays (biofilm, sporulation and seed colonization) as well as our first in vivo (root colonization) assay were all developed to quantify the colonization potential of our recombinant B. subtilis. These assays confirm that the strains don’t have any potential defects due to the Cyt proteins being highly expressed. Then the time-course cytotoxicity assay is aimed at providing clearer data of rate of killing over time and examining how long it takes for 100% of the flies to die and quantifying the insecticidal activity of the B. subtilis strains under ideal conditions. Finally, the greenhouse field trial would be the final experiment where we validate the system in situ to quantify how well our B. subtilis strains can protect tomato plants against an infection with fungus gnats.

Investigating B. subtilis-Cyt mutants biofilm formation capacity

Biofilms are communities of microbes that attach to surfaces, which can be found in medical, industrial and natural settings (O'Toole, 2011). These structures adhere to surfaces with the help of extracellular polymeric substances (EPS), and are formed by B. subtilis. As an endophyte, B. subtilis is highly effective at colonizing plant roots, where these biofilms act as physical barriers, providing a method of protection for plants from a range of factors such as pathogens, extreme pHs and temperatures from their external environments (Wang et al., 2019).

To ensure that our recombinant B. subtilis strains retained their ability to form biofilms, our team adapted a biofilm accumulation experiment from O’Toole (2011). In short, this method used a 96-well microtiter plate to analyze microbial biofilm formation, in which overnight cultures of each strain of B. subtilis (B. subtilis WT, B. subtilis pCG004-GFP, B. subtilis pCG004-Cyt1Aa, B. subtilis pCG004-Cyt2Ba) were supplemented with 20% L-arginine and 20% glycerol to promote biofilm formation. The cells were washed with PBS and bacterial adherence to wells due to biofilm formation were visualized through crystal violet staining. The concentration of crystal violet in each well is proportional to the number of cells in the biofilm. For measurement by spectrophotometry, we measured absorbance at 550 nm which reflected the biofilm formation of the four tested B. subtilis isolates (O'Toole, 2011).
The untransformed WT strain of B. subtilis, and the three transformed strains of B. subtilis (Empty Backbone, Cyt1Aa, and Cyt2Ba) were incubated in Tryptic Soy Broth for a span of 24 hours, which was then read through a microplate reader (Table 2).

The OD550 values were all normalized to the average of the WT values (Table 3) and plotted on a scatter plot (Figure 1).

Figure 1. B. subtilis WT, Empty Backbone, Cyt1Aa and Cyt2Ba were grown in Tryptic Soy Broth and incubated for 24 hours in a 96-well plate before being washed and stained with crystal violet, followed by an absorbance reading at 550 nm. The figure represents normalized values of the OD550 when compared to the WT control and each value was plotted finding the mean from eight replicates tested. The Standard Error of the Mean (SEM) was calculated and shown in the error bars. Strain names can be found in Table 1 and the raw data for this graph can be found in Table 3.

From the data seen in Figure 1, it can be seen that biofilm formation capabilities were not significantly impacted by fluorescent genes encoding GFP, BFP and mScarlet, as well as cytotoxic proteins to WT B. subtilis. This indicates that our B. subtilis-cyt strains are able to retain their natural ability to form a biofilm and thus colonize roots.

Sporulation Testing

One of the strategies B. subtilis uses for efficiently surviving environmental challenges includes producing spores in response to extreme stress like nutrient depletion, and pH, temperature, and pressure changes. The bacterial genome is safe inside the spore until environmental conditions improve, in which the spore leaves the state of dormancy into a vegetative state (McKenney et al., 2013). By purifying B. subtilis spores, we can ensure that our biopesticide can be stored for global distribution in an inert form with a long shelf-life. Once they’ve arrived at their destination, seeds can then be coated with our spore suspension by growers for simple distribution and colonization. As an endophyte, B. subtilis is considered to be a plant growth-promoting bacteria (PGPB), which can provide plants with protection from biotic and abiotic stresses, and enhances plant growth and performance by providing the seedlings with nutrients and protection (Fiorenzano et al., 2017).

We used a sporulation protocol provided to us by iGEM Frieburg 2016. In summary our team grew the B. subtilis strains in Difco Sporulation Medium (DSM), and conducted a lysozyme treatment to kill vegetative cells necessary for spore purification. The purified spores of B. subtilis were then analyzed using Sony FACS for flow cytometry (Figure 2).

WT B. subtilis was the negative control that did not undergo a lysozyme treatment, in which the set gate showed that 39.77% of the sample consisted of spores. After purification, the amount of spores in each sample slightly increased, with an average of 56.04% spores in all of the mutant samples. Overall there was no significant difference in the number of spores produced among the strains tested.

Seed coating

Unfortunately iGEM Guelph did not have time to complete this assay. However, the original assay was intended to function by coating our tomato seeds with our purified spores, and conduct a germination experiment to assess if seeds coated in our transformed strains of B. subtilis can grow in 1% agar and has the ability to kill fungus gnat larvae.

Cytotoxicity Bioassay

This assay is identical to the cytotoxicity assay carried out in Engineering Success however, more detail will be added here as we intend to expand the parameters of this assay. Our team conducted an attractive-toxic sugar bait (ATSB) toxicity bioassay testing the effects of cyt proteins Cyt1Aa and Cyt2Ba on the order diptera, using Drosophila melanogaster as a model. ATSB systems are ideal for toxicity assays, as they combine an attractive substance for the organism, with the toxin being used (Maia et al 2018). Upon our system’s activation by IPTG, Cyt proteins are produced by B. subtilis, and ingested and solubilized into a protoxin in the mid-gut of the insect which has an alkaline pH (Keeton et al. 1998). These protoxins are cleaved by species-specific proteases and bind to mid-gut membrane receptors, signalling apoptosis (Keeton et al. 1998). Our toxicity bioassay aims to understand the effect that Cyt1Aa and Cyt2Ba have on dipteran mortality, which will give the team an indication of how our product could work in real-world scenarios.

As shown in Engineering Success to determine the effect that each strain had on mortality of D. melanogaster, an ATSB system was used. It was observed that no death occurred in the period of 20 minutes for the WT strain of B. subtilis. Compared to the WT strain, 11% of the organisms were found to be deceased at the 20-minute time point for our transformed GFP strain (B. subtilis pCG004) control. Higher rates of mortality were observed for our transformed B. subtilis strain containing the cyt proteins Cyt1Aa and Cyt2Ba, at 67% and 71%, respectively. Our transformed E. coli strains containing the Cyt1Aa and Cyt2ba proteins also demonstrated high mortality rates at 43% and 47%, respectively, although the rate is lower than in B. subtilis (Figure 3). On the other hand, we had done preliminary attempts of the toxicity assay which were identical to the ones described, except we did not add IPTG. No death of flies was observed in the period of 20 minutes when cytotoxin expression was not induced by IPTG.
Protocols were adapted from Wongthangsiri et al (2018) and Fiorenzano et al (2018). We chose to use wingless D. melanogaster (mutant defective in vestigeal gene) as they are easier to manage in our assay. To conduct the toxicity bioassay, an ATSB system was used, combining an overnight culture of each strain (WT 1A976 B. subtilis, B. subtilis pC004 control, B. subtilis pC004-Cyt1Aa, B. subtilis pC004-Cyt2Ba) with a volume of 50% sucrose solution that would dilute the culture to an OD of 0.8 (or approximately 109 cells). Flies were moved using an aspirator to individual plastic containers to ensure no outward variables impacted the results. For our work in Proof of Concept, three technical replicates were used per strain, with approximately 5 flies acting as biological replicas per container. Flies were exposed to halved cotton pads with the ATSB solution for a total of 30 minutes. Mortality was observed every 5 minutes. For this, we expect to create a Kaplan-Meyer curve similar to Figure 3 with more detail on death rates at prior timepoints and the final time that causes 100% killing. After re-doing the experiment we obtained the results seen in Figure 4.

Root colonization

This protocol would have been used to examine biofilm formation on the roots of Beefsteak tomato plants, one of the most common greenhouse grown tomato plants in Ontario. Tryptic Soy Agar (TSA) will be the general growth medium used for the isolation and cultivation of Bacillus subtilis. Roots will be sterilized by submerging them in a beaker with 70% ethanol, following a treatment with a 0.3% sodium hypochlorite solution prior to plating. After roots are plated, a bacterial suspension of WT 1A976 B. subtilis, B. subtilis pC004, B. subtilis pC004-Cyt1Aa, B. subtilis pC004-Cyt2Ba overnights would be separately introduced to the TSA plate and incubated for a span of 24 and 48 hours. We hoped to visualize the effects of our transformed B. subtilis strains through confocal microscopy on biofilm formation.

This experiment has a great possibility of allowing us to use our transformed B. subtilis strains as a biostimulant, which can be further proven by conducting a soil drenching experiment, potentially in a phytotron or greenhouse environment. Soil drenching is the introduction of a bacterial suspension to a plant through means of root dipping or soil amendment to promote soil health and enhance root development (Mofokeng et al., 2021). As one of the products we aim to put out into market are coated seeds, being able to provide growers and farmers with another method of application can further prove the effectiveness of iGEM Guelphs work this year.

References

  • O'Toole G. A. (2011). Microtiter dish biofilm formation assay. Journal of visualized experiments : JoVE, (47), 2437.
  • Yin, W., Wang, Y., Liu, L., & He, J. (2019). Biofilms: The Microbial "Protective Clothing" in Extreme Environments. International journal of molecular sciences, 20(14), 3423.
  • McKenney, P. T., Driks, A., & Eichenberger, P. (2013). The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nature reviews. Microbiology, 11(1), 33–44.
  • Rocha, I., Ma, Y., Souza-Alonso, P., Vosátka, M., Freitas, H., & Oliveira, R. S. (2019). Seed Coating: A Tool for Delivering Beneficial Microbes to Agricultural Crops. Frontiers in plant science, 10, 1357.
  • Fiorenzano, J. M., Koehler, P. G., & Xue, R. D. (2017). Attractive Toxic Sugar Bait (ATSB) For Control of Mosquitoes and Its Impact on Non-Target Organisms: A Review. International journal of environmental research and public health, 14(4), 398.
  • Wongthangsiri, D., Pereira, R. M., Bangs, M. J., Koehler, P. G., & Chareonviriyaphap, T. (2018). Potential of attractive toxic sugar baits for controlling musca domestica L., drosophila melanogaster meigen, and Megaselia scalaris Loew adult flies. Agriculture and Natural Resources, 52(4), 393–398.
  • Maia, M.F., Tenywa, F.C., Nelson, H. et al. Attractive toxic sugar baits for controlling mosquitoes: a qualitative study in Bagamoyo, Tanzania. Malar J 17, 22 (2018).
  • Keeton, T. P., Francis, B. R., Maaty, W. S., & Bulla, L. A., Jr (1998). Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins. Applied and environmental microbiology, 64(6), 2158–2165.
  • Mofokeng, M. M., Araya, H. T., Araya, N. A., Makgato, M. J., Mokgehle, S. N., Masemola, M. C., … Amoo, S. O. (2021). Integrating biostimulants in agrosystem to promote soil health and plant growth. Biostimulants for Crops from Seed Germination to Plant Development, 87-108.
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