Experiments

Experiments

C.R.O.P.S Detection Kit Design

The C.R.O.P.S detection kit for clubroot is designed to provide farmers with a simple, reliable, and user-friendly method to detect the presence of Plasmodiophora brassicae in soil samples. By utilizing antibody fragments like nanobodies and FABs (Fragment Antigen Binding), our detection kit offers a high degree of specificity while maintaining ease of use for non-specialized operators. This year's design features improvements in protein purification and solubility to enhance performance in a lateral flow assay (LFA), pivoting from our previous reliance on Direct ELISA.

Nanobody Design

Our engineered nanobody incorporates several key features to ensure effective binding and easy purification:

  • 6x Histidine Tag: This tag facilitates nickel affinity chromatography, simplifying the protein purification process, ensuring high yield and purity of the nanobody.
  • Poly-lysine Tag: This was added to ensure efficient binding of the fluorescent probe used in our detection experiments. The tag allows chemical binding of gold nanoparticles to lysine residues, making the nanobody adaptable for both ELISA tests and LFAs.
  • ompA Tag: To improve the solubility of the nanobody, we added an ompA tag. This tag enhances overexpression and solubility, making the nanobody more stable and easier to produce. Our team’s past success using this tag on similar proteins supports its reliability for this project.

This design is intended to allow for rapid binding to the target antigen, ensuring the detection system remains sensitive and reliable in field conditions.

FAB Design

The FAB fragment is composed of two domains (light and heavy chains), each with their own specialized tags to optimize functionality:

  • 6x Histidine Tag (Heavy Chain): Similar to the nanobody, this tag is utilized to simplify protein purification through nickel affinity chromatography.
  • Poly-lysine Tags (Light and Heavy Chains): Both chains are tagged with poly-lysine to ensure efficient binding of fluorescent probes and gold nanoparticles, making the FAB fragment suitable for ELISA and LFAs.
  • Solubility Tags (ompA and phoA): To enhance solubility and overexpression, ompA and phoA tags were added to the light and heavy chains, respectively. These solubility tags were chosen based on our team's previous success using them in related experiments.

The dual nature of the FAB fragment increases the robustness of antigen detection while maintaining a monovalent binding characteristic to minimize non-specific interactions.

Transition from Direct ELISA to Lateral Flow Assay (LFA)

The 2023 ULethbridge iGEM Team used a direct ELISA method to validate the binding efficiency between their chimeric GFP protein (COAPE-GFP) and the Plasmodiophora brassicae antigen pbEL04. While effective in a controlled laboratory setting, the direct ELISA process requires specialized equipment and technical expertise, limiting its applicability for on-site use by farmers.

In response to this limitation, we have pivoted from using direct ELISA to the development of a lateral flow assay. LFAs offer several advantages:

  • Ease of Use: LFAs are straightforward and can be performed without specialized equipment, making them ideal for use in the field.
  • Rapid Results: Results from an LFA can be obtained within 5-20 minutes, allowing for quick decision-making in agricultural settings.
  • Scalability: LFAs are cost-effective and can be easily scaled for mass production and widespread use.

This year’s transition ensures that our detection kit will be more accessible to farmers, allowing them to perform tests on-site with minimal training. The incorporation of gold nanoparticles in the LFA increases visual clarity of the results, ensuring that users can easily interpret the presence of Plasmodiophora brassicae in their soil samples.

Future Considerations

Our design strategy will leverage well-established biochemical methods like nickel affinity chromatography and gold nanoparticle binding to create a detection kit that is both reliable and scalable. Future developments will focus on optimizing the sensitivity and specificity of the lateral flow assay, as well as testing in real-world agricultural conditions to ensure efficacy across diverse soil types.

C.R.O.P.S Biopesticide Design

Our biopesticide strategy aims to harness the synergistic capabilities of Bacillus subtilis and Pseudomonas fluorescens to combat clubroot disease. By engineering these microorganisms, we can enhance their natural biocontrol properties, providing a sustainable and effective solution for agricultural applications. This section details the design of the constructs used to boost the production of antifungal compounds and plant immune response molecules, helping protect crops from fungal and fungal-like pathogens like Plasmodiophora brassicae.

DegQ Expression Construct

The DegQ Expression Construct is designed to enhance the production of fengycin, a lipopeptide with potent antifungal properties, through the introduction of the degQ gene from Bacillus subtilis XF-1. Fengycin plays a critical role in suppressing fungal and fungal-like pathogens, which makes this construct particularly useful for biocontrol applications.

Key Components:

  • T7 Promoter (BBa_I719005): A strong, inducible promoter used in E. coli systems that express T7 RNA polymerase. This ensures high-level transcription of the degQ gene, maximizing fengycin production.
  • RBS (BBa_B0034): A widely used ribosome binding site that ensures efficient translation of the degQ gene, enabling high levels of protein production.
  • degQ Gene: The core component of the construct, encoding a regulatory protein that enhances the production of fengycin by increasing the phosphorylation of DegU (DegU-P). This, in turn, activates the expression of genes responsible for producing extracellular enzymes such as proteases, amylases, and cellulases, all of which contribute to pathogen cell wall degradation in the soil.
  • T1 Terminator (BBa_B0010): A terminator sequence that halts transcription efficiently, ensuring the stability and proper processing of the mRNA.

The DegQ construct ensures high expression levels of the degQ gene, which enhances the biocontrol properties of Bacillus subtilis by boosting the production of fengycin and other pathogen-degrading enzymes. This contributes to a reduction in the soil pathogen load, helping to control clubroot disease in agricultural settings.

Native DegQ Expression Construct

The Native DegQ Expression Construct uses the natural regulatory elements of the degQ gene, allowing for controlled expression that mimics physiological conditions. This construct is designed for use in systems where maintaining the native expression patterns of DegQ is important for studying its role in biocontrol.

Key Components:

  • PdegQ Promoter (BBa_K3429006): The native promoter for the degQ gene, ensuring that the gene is expressed in response to the same environmental cues and regulatory factors as in its natural host.
  • RBS (BBa_B0034): This ribosome binding site ensures that translation occurs efficiently, maximizing protein production.
  • degQ Sequence (BBa_K5091005): The native coding sequence for the degQ gene, which enhances the production of fengycin and regulates the expression of extracellular enzymes involved in pathogen control.
  • DegQ Terminator (BBa_K3429007): A native terminator sequence that ensures proper transcription termination, increasing mRNA stability and contributing to efficient protein production.

This construct is ideal for maintaining native expression levels of the degQ gene, offering an approach that more closely resembles the gene's natural regulation while still enhancing fengycin production for biocontrol.

pmsC-pmsB Expression Construct

The pmsC-pmsB Expression Construct co-expresses two genes from Pseudomonas fluorescens that are essential for the biosynthesis of salicylic acid (SA), a critical component of the plant’s immune response. Salicylic acid enhances systemic acquired resistance (SAR), helping plants protect themselves from a wide range of pathogens, including Plasmodiophora brassicae.

Key Components:

  • araC-pBAD Promoter (BBa_I0500): An arabinose-inducible promoter that allows tight regulation of gene expression. This ensures that pmsC and pmsB are only expressed when needed, reducing metabolic burden on the host before induction.
  • RBS (BBa_B0030 & BBa_B0034): Strong ribosome binding sites used for efficient translation of both the pmsC and pmsB genes, ensuring high levels of protein synthesis.
  • pmsC Gene (BBa_K5091008): Encodes isochorismate synthase, which catalyzes the first step in the SA biosynthesis pathway. Isochorismate is the precursor to salicylic acid.
  • pmsB Gene (BBa_K5091009): Encodes isochorismate pyruvate lyase, which converts isochorismate into salicylic acid, completing the biosynthetic pathway.
  • Terminator (BBa_B0012): A double terminator that ensures efficient termination of transcription, preventing mRNA degradation and ensuring stable protein production.

The pmsC-pmsB construct enhances salicylic acid production in Pseudomonas fluorescens, boosting the plant’s natural defenses. This increases the plant’s resistance to pathogens by promoting the SA-mediated immune response, providing long-lasting protection against infections.

Synergistic Biocontrol Strategy

By co-deploying Bacillus subtilis and Pseudomonas fluorescens in a biopesticide formulation, we could leverage the complementary mechanisms of these microorganisms. The degQ gene boosts the production of antifungal compounds like fengycin in Bacillus subtilis, while the pmsC-pmsB construct enhances the production of salicylic acid in Pseudomonas fluorescens. Together, these modifications provide a dual approach to biocontrol:

  • Fengycin production directly attacks fungal and fungal-like pathogens in the soil, reducing their ability to infect plants.
  • Salicylic acid boosts the plant's immune response, increasing its resistance to subsequent pathogen attacks.

This synergistic approach provides a comprehensive, sustainable solution for managing clubroot disease, reducing the need for chemical fungicides and promoting healthier crop yields.

Note on Project Focus

Due to limited resources and time constraints, our team decided to focus our wet lab and dry lab efforts on the development of the detection kit. While the biopesticide design holds significant promise, we prioritized the detection kit as a more feasible and impactful solution within the scope of this year's project.

PDF Document of Our Experiments

References

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Shengye Guo, Xingyu Li, Pengfei He, Honhing Ho, Yixin Wu, Yueqiu He, Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological control and multiple beneficial properties in plants, Journal of Industrial Microbiology and Biotechnology, Volume 42, Issue 6, 1 June 2015, Pages 925–937, https://doi.org/10.1007/s10295-015-1612-y (opens in a new tab)

Xuefei Jiang, Ying Su, Maolin Wang et al. A small cysteine-rich protein identified from the Proteome of clubroot pathogen, Plasmodiophora brassicae, induces cell death in nonhost plants and host plants, 29 August 2022, PREPRINT (Version 1) available at Research Square https://doi.org/10.21203/rs.3.rs-1961445/v1 (opens in a new tab)

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