Compared with traditional chemical pesticides, biopesticides have lower risk of environmental pollution and less toxic side effects, which have less impact on human body and environment. Therefore, we focused on the development of new biopesticides in the early stage of brainstorming, hoping to provide new inspiration for this.

After our research, we found that Bacillus subtilis is a more widely used engineering bacteria in biopesticides. We obtained a specification for a biopesticide using Bacillus subtilis as a biological chassis and designed a Bacillus subtilis-based biopesticide based on it.

Targeting - Enrichment

(1) Strong motility using the natural chemotaxis ability of engineering bacteria targeting, do not have to design.

(2) Enrichment at infestation

Advantage:

(1) Compared with the spore display technique, steps such as spore purification are eliminated and the cost is lower.

(2) It can take full advantage of the strong motility of the bacteria itself to enhance the targeting ability.

The Agr system of Staphylococcus aureus (S. aureus) was chosen as the module for signal emission and reception, consisting of an auto-inducing peptide (AIP), a receptor (AgrC), and a transcription factor (AgrA). AIP is generated and secreted by AgrD, AgrB (BBa_K3447010) When the number of transgenic Bacillus subtilis increases to a certain level, AIP signaling accumulates, and AgrC senses the signaling molecule and phosphorylates AgrA, activating the expression of promoters P2 (BBa_I746104), P3 (BBa_K212003).

Functional module

Expression of asparagus antimicrobial peptide GAFP-1, synthesized antimicrobial peptide D4E1 (BBa_K1364013), secreted through SamQ signal peptide (BBa_K1074014), and connected by protein junction (BBa_K2934005).

Figure 4: The functional module I

Via isobranchial acid pyruvate lyase and isobranchial acid synthase (pchBA, BBa_J45017); converts branchial acids to salicylic acid (SA) and pyruvic acid, the former of which induces systemic acquired resistance (SAR) in plants.

Figure 5: The functional module II

Biosafety module

(1) The chassis strain of Bacillus subtilis is a common soil microorganism and plant symbiotic bacteria, which itself will not cause greater damage to the ecological environment.

(2) The designed functional genes need to be activated by group induction after the enrichment of two strains at the same time in the pathogenic bacteria infestation, which will limit the proliferation of engineering bacteria and the impact on the ecology to a certain extent.

(3) The selected antimicrobial peptides should be biological components existing in nature, or modified and designed for plant pathogenic fungi, with the action site in the cell wall of the fungus, which is harmless to human beings and plants.

(4) Enhance the stability of the system through the use of integrating plasmids to limit horizontal gene transfer.

(5) Add MazEF toxin antitoxin system (BBa_K302032), add the toxin gene MazF gene within the gene of the modified module, and place the antitoxin gene MazF in a more distant position to reduce the possibility of horizontal gene transfer.

Figure 6: The biosafety module

As a result of our analysis, we have summarized the feasibility and shortcomings of our project as follows:

We endorsed the idea of biopesticide development, but with a deeper consideration of the chassis. We began to read more literature in an effort to keep our thinking from being limited by the biopesticide products currently available.

We started reading more literature and came across one that provided the original inspiration for our flora sentinel. Researchers from the University of East Anglia in the UK have devised a novel approach to enhance plant disease resistance using animal antibodies. However, the current ID sequence lacks tight binding and specificity. Therefore, our proposed solution involves substituting the ID sequence with a specially engineered nanobody capable of precise effector binding, while still retaining the ability to recruit signaling NLR and trigger downstream immune responses.

Figure 7: The biosafety module

Our design is to replace the ID sequence with a nanobody that specifically binds to effects by directly replacing the ID sequence fragment (base 509-730) of pickm-1 with enhancer, and trying to retain its ability to repeat signaling NLR and activate downstream immediate responses after modification.

We successfully modified a natural plant immune receptor (NLR), and the method is to replace its original ID sequence (the original pathogen-recognizing sequence in plant) with a nanobody that specifically recognizes the effector. This modified NLR would improve plant’s ability to recognize pathogenic bacteria while retaining original NLR’s ability to stimulate downstream immune responses, thereby enhancing the resistance of plants to pathogenic bacteria. Please click on our Result for more information.

Our future work will focus on improving the binding specificity of nanobodies and expanding their recognition of different effectors. By optimizing and improving the nanobodies, we can ensure that it binds tightly and specifically to the desired effector molecules, thereby enhancing plant defense against pathogens. In addition, expanding the range of effectors that can be recognized by engineered nanobodies will further enhance the immune response of the plant and improve the overall disease resistance.

We have already optimized and improved the nanobodies through computer technology by means of bioinformatics analysis and developed user-friendly software to assist our biopesticide products. In the future, there is hope that targeted nano-antibodies can be designed for any pathogenic bacteria through computer technology, which could be a good solution to the problem of pathogen resistance caused by conventional pesticides.

Our initial inspiration for this module came from the article "tRNA-Related Sequences Trigger Systemic mRNA Transport in Plants". In this article, the researchers found that mRNAs containing tRNA-like structures (TLSs) were enriched in the phloem stream and could move across graft junctions.

Therefore, we would like to introduce TLS module into the project, equipped our RNA with the ability to transport in plant vascular tissues.

For early screening, we obtained 20 tRNA sequences of Arabidopsis thaliana from the ncbi database, and performed RNA secondary structure prediction, using tRNA containing the above structure for subsequent experiments.

We utilize phenotypic observation to assess the functionality and mobility of the transported mRNAs.

To further validate the functionality of the TLS module, we can obtain additional tRNA sequences from different species and perform RNA secondary structure prediction on them. By testing these additional TLS elements, we can select the elements that perform best in mRNA transport across plant vascular tissues.

Based on our initial screening of 20 tRNA sequences from Arabidopsis thaliana, we have selected a set of TLS elements for further experimentation. Additionally, we expanded our analysis to include tRNA sequences from different plant species to maximize the diversity of TLS elements.

The next phase of our project will involve experimental validation of the selected TLS elements.

To verify the TLS-triggered mobility of fused transcript in vascular bundle of Nicotiana tabacum，we amplified EGFP gene from plasmid pcDNA3.1-T2A-EGFP. 20 tRNA sequences of Arabidopsis thaliana and Nicotiana tabacum are synthesized. Then 4 plasmids pGREENII-GFP(-TLS) is constructed, which contains 35S promoter and T-DNA repeat to efficiently express our mRNA in plant cell.

We judged the intensity of protein expression by observing the amount of leaf fluorescence expression, but we did not get a better observation because of the low intensity of fluorescence expression and the high interference of experimental observation. Therefore, we began to explore new characterization modes.

We will explore new characterization methods to improve the observation and quantification of protein expression levels. This will allow us to better understand the effects of TLS elements on gene expression and manipulation in plants.

We wanted to characterize the mobility of TLS by phenotypic observations, qPCR and protein expression assays. In terms of phenotypic observation, we have constructed a total of three expression systems, GFP, GUS, and RUBY, by reviewing literature to explore the best phenotypic expression effect.

We conducted qPCR experiments to confirm the movement of TLS-triggered GFP mRNA in plants. The results indicated that mRNA containing TLS had moved further away from the injection site.

Additionally, the researchers performed GUS staining and used RUBY to visualize the movement and expression of mRNA in plant cells. The results revealed that the mRNA sequence containing the Ala TLS was able to move within the leaves, as evidenced by the presence of blue GUS catalytic products in the vascular bundles.

See more details in Result.

Further analysis and experimentation are needed to gain a deeper understanding of TLS mobility and its implications in plant biology. This study provides a foundation for future research in this area, and continued investigation may lead to valuable insights into the mechanisms and potential applications of TLS elements in plants.

Our engineered strains are mainly used in plants, and plants need sunlight to grow, so we chose sunlight as the way to initiate suicide. We began screening and researching toxic proteins to select the most appropriate ones for the biosafety module.

We initially considered KillerRed (BBa_K1184000). According to extant research, KillerRed is a red fluorescent protein that generates reactive oxygen species (ROS) under yellow-green light (540-585 nm). Given that plants typically absorb blue-violet and red light, reflecting light near 540nm (green), KillerRed is ideally suited as a biosafety 'kill switch'.

Other proteins in the KillerRed family, such as Supernova, were also validated. It has been shown to reduce dimerization tendency and maintain the ability to generate ROS. We obtained its fragment by point mutation, but light exposure showed that its suicide inducing effect was less stable than that of KillerRed.

Figure 9: Results of light experiments of Agrobacterium with KillerRed V.S. empty plasmid (on a sunny day)

In order to ensure the success and reliability of our biosafety module, we deemed it necessary to explore additional protein options. By switching to a different protein, we hope to find one that not only demonstrates stability in inducing suicide but also aligns with the specific needs and requirements of our engineered strains in plants.

This decision to replace Supernova serves as an important learning experience for future research and development projects. The stability and consistency of the chosen protein should be thoroughly tested and validated before implementation, ensuring that it can effectively execute the desired actions in the given context.

By reading more literature and consulting with more senior researchers, we identified another phototoxic protein.

By reading more literature and consulting with more senior researchers, we identified another phototoxic protein.

our plate light experiment demonstrated a noticeable reduction of engineered bacteria under natural light irradiation, regardless of whether the conditions were sunny or cloudy.

Figure 10: Results of light experiments of Agrobacterium with empty plasmid V.S. minisog (On a cloudy day)

We have gained knowledge about the significance of considering the specific needs of the target organism and the importance of rigorous testing and validation. Moving forward, we can use this experience to improve our designs and expand our understanding of biosafety measures in genetically engineered organisms.