- To obtain the nucleotide sequences of our parts and,
- To assemble Level 0 plasmids.
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Table of Contents
Laboratory / Design and Engineering
Design and Engineering
Following a systematic Design-Build-Test-Learn approach to develop robust biocontrol solutions through iterative engineering cycles. Close the side bar for best visualization!
Cycle 1. Preparation of the new parts in the SubtiToolKit syntax
Cycle 1 focuses on the implementation of new basic parts compatible with the SubtiToolKit syntax.
Engineering Iterations
Design
SubtiToolKit as a tool for modular assembly
Based on our literature review, we selected SubtiToolKit (STK) as the best potential tool for the engineering of B. subtilis [1]. STK is a collection of parts optimized for their use in B. subtilis, allowing to assemble constructs – replicative or integrative plasmids – in a one-pot, Golden Gate reaction. Thanks to the kind support of prof. Joaquin Caro-Astorga, we acquired a 96-well plate with different DNA parts, which we could then use to assemble our final constructs.
Figure 1: SubtiToolKit (STK) assembly workflow showing the systematic approach from Level 0 entry vectors to Level 1 functional constructs through Golden Gate assembly.
Build
Subtitle
We used GenSmart™ Codon Optimization tool [4] to re-design the nucleotide sequences of the chosen AMPs. The following restriction sites were avoided in the design: BsaI (required for the cloning of type 1 plasmids), BbsI (required for the cloning of level 0 plasmids), SapI (RFC1000 compatibility requirement), BsmBI (required for the cloning of level 2 plasmids).
Next, we added appropriate overhangs to the re-designed sequences (Tab.1). In this form, sequences were ordered in the form of gene fragments from IDT or Twist.
After resuspension of the gBlocks according to the producer's requirements, parts underwent Golden Gate Assembly (GGA) with pSTK-0-GFP entry plasmid using BbsI restriction enzyme and T4 ligase (New England Biolabs). Initially, we used the amalgamation of the protocols described by [1] and New England Biolabs, where the gBlock and the plasmid were mixed in 2:1 molar ratio, and the reaction consisted on 20 cycles – 1 min digestion in 370C and 1 min ligation in 160C with a final digestion step – 20min 600C.
Next, we used primers designed by the authors of STK to identify positive clones after transformation of the GGA mixtures to DH10b competent cells. First, non-green colonies were selected utilizing the blue-light transilluminator. The reaction conditions were as described in (Link: Golden Gate Assembly – type 0 vectors (BbsI) in the Experiments page). All chosen colonies were negative – amplified DNA fragments were the size of the GFP dropout cassette. The next day, re-plated colonies grew with a green tint, confirming that they do not contain phoA coding sequence, but GFP.
Test
Validation & Characterization
Comprehensive validation through laboratory characterization, performance metrics, and functional assays under controlled and field-like conditions.
- Functional bioactivity testing protocols
- Performance metrics evaluation and benchmarking
- Environmental stability and stress testing
- Safety assessment and toxicity evaluation
Figure 3: Field testing environment showing cacao trees where biocontrol systems undergo comprehensive validation and performance assessment.
Rigorous Validation Framework
Our comprehensive testing framework encompasses both individual component validation and integrated system performance assessment under carefully controlled laboratory conditions. The validation process is designed to generate reliable, reproducible data that supports regulatory submissions and informs commercialization decisions.
Functional bioactivity testing employs standardized assays to quantify antimicrobial efficacy against target pathogens. These tests include minimum inhibitory concentration (MIC) determinations, time-kill kinetics, and resistance development studies conducted under various environmental conditions that mirror real-world applications.
Environmental stability testing evaluates system performance under diverse conditions including temperature variations, pH fluctuations, and exposure to agricultural chemicals. This comprehensive assessment ensures reliable performance across different geographical regions and seasonal conditions.
Safety evaluation includes comprehensive toxicity assessments, environmental impact studies, and risk analysis protocols. These studies follow international guidelines and regulatory frameworks to ensure safe deployment and minimize potential adverse effects on non-target organisms and ecosystems.
Learn
Subtitle
Low efficiency was linked to BbsI's slower activity, so steps were separated and part design and reagent quality were checked.
After discussions with our PI, we saw that a potential reason for the seemingly low efficiency of our reaction might be a suboptimal time of ligation and restriction incubations. Based on the experience of the lab, BbsI is thought to be less efficient than other Type IIS enzymes, and it might require longer incubation time. On the other hand, we expect the ligation of the two fragments to be quite efficient. Thus, instead of cycling between restriction and incubation steps in a one pot reaction, we separated the reaction steps.
Additionally, we needed to explore the possibility that problems might be related to our part design or the quality of reagents.
Cycle 2. Set up of a secretion assay in B. subtilis
In cycle 2, we assembled constructs encoding for fusions of a reporter protein PhoA with secretion tags, and evaluated them in an optimized assay.
Engineering Iterations
Design
Advanced System Architecture & Optimization
Enhanced system architecture incorporating lessons learned from Cycle 1, with focus on scalability, efficiency, and production readiness.
Figure 5: Advanced system architecture design incorporating scalability improvements and production-ready specifications for commercial deployment.
Advanced Design Strategy - Cycle 2
Building upon the foundational work from Cycle 1, this advanced design phase focuses on optimization and scalability improvements that prepare the PhytoBlock system for commercial deployment and real-world applications.
The enhanced architectural design process incorporates lessons learned from initial prototyping, focusing on creating robust, scalable systems that can be efficiently manufactured and deployed at commercial scale while maintaining system performance and reliability.
Advanced computational modeling and machine learning approaches are employed to optimize system performance, predict potential bottlenecks, and design preventive solutions that ensure consistent operation across diverse deployment conditions.
Production-ready design specifications incorporate cost optimization strategies, manufacturing constraints, and regulatory compliance requirements to ensure smooth transition from research prototype to commercial product.
Build
Optimized Construction & Scale-up
Advanced manufacturing processes and scale-up protocols for enhanced production efficiency.
- Automated production protocols
- Quality control optimization
- Scale-up manufacturing processes
- Cost reduction strategies
Figure 6: Optimized manufacturing processes showcasing automated production protocols and advanced quality control systems for scalable implementation.
Advanced Manufacturing Strategy - Cycle 2
Implementation of optimized construction methods with focus on scalability, reproducibility, and cost-effectiveness for commercial production. This phase incorporates automated manufacturing processes and advanced quality control systems.
Production scaling involves systematic optimization of manufacturing workflows, implementation of continuous monitoring systems, and development of robust supply chain management protocols that ensure consistent product quality across different production batches.
Advanced automation technologies including robotics, process control systems, and real-time quality monitoring are integrated to minimize human error, reduce production costs, and maintain consistent manufacturing standards.
Quality assurance protocols include comprehensive testing at each production stage, statistical process control implementation, and traceability systems that enable rapid identification and resolution of any production issues.
Test
Enhanced Validation & Performance Testing
Comprehensive testing protocols for optimized system performance and reliability assessment.
- Performance benchmarking studies
- Stability and shelf-life testing
- Field trial protocols
- Regulatory compliance validation
Figure 7: Enhanced validation protocols demonstrating comprehensive field trials and performance benchmarking for regulatory compliance and market readiness.
Advanced Testing Framework - Cycle 2
Advanced testing methodologies to validate improvements and ensure consistent performance across different conditions and scales. This comprehensive framework includes accelerated stability testing, real-world simulation protocols, and statistical validation procedures.
Performance validation encompasses extensive field trials under diverse environmental conditions, long-term effectiveness studies, and comparative analysis against existing solutions to demonstrate superior performance characteristics.
Regulatory compliance testing follows international standards and guidelines, including comprehensive safety assessments, environmental impact evaluations, and efficacy demonstrations required for market approval and registration.
Advanced analytical techniques including high-throughput screening, real-time monitoring systems, and predictive modeling enable comprehensive performance characterization and optimization recommendations for continued improvement.
Learn
Optimization Analysis & Scaling Strategy
Analysis of optimization results and development of scaling strategies for commercial deployment.
- Performance improvement analysis
- Commercial viability assessment
- Scaling strategy development
- Market deployment planning
Figure 8: Strategic analysis framework utilizing advanced analytics and market intelligence for commercial deployment planning and scaling strategy development.
Strategic Analysis & Planning - Cycle 2
Comprehensive evaluation of improvements achieved and strategic planning for large-scale deployment and commercialization. This phase involves detailed market analysis, competitive positioning, and strategic partnership development for successful market entry.
Advanced data analytics and machine learning techniques are employed to extract actionable insights from comprehensive testing data, customer feedback, and market research to optimize product positioning and commercial strategy.
Strategic planning encompasses global market expansion strategies, regulatory pathway optimization, and intellectual property protection to ensure sustainable competitive advantage and long-term market success.
Continuous improvement frameworks are established to enable ongoing product optimization, customer feedback integration, and adaptive strategy development that ensures sustained market leadership and customer satisfaction.
Cycle 3. Set up of a plate-based dual culture assay
In this cycle, we optimize the dual-culture assay conditions by measuring the microbial growth in different media and temperatures .
Engineering Iterations
Design
Deployment-Oriented Architecture
Iteration 1 expands architecture for robust field deployment with telemetry, resilience and maintainability layers.
- Deployment configuration matrices
- Resilience & redundancy planning
- Telemetry & traceability hooks
- Field biosafety safeguards
Design enhancements focus on operational observability, modular update pathways, and fault containment boundaries suitable for scale.
Build
Field-Ready Assembly
Assembly emphasizes reproducibility, containerization concepts (where applicable) and rapid rollback.
All constructs verified across environmental parameter brackets to ensure stability prior to deployment runs.
Test
Stress & Field Simulation
High-intensity validation incorporating environmental stress, failure injection and longitudinal performance tracking.
Test matrix covers temperature, pH, humidity, competitive microbiome exposure, and transport shock simulations.
Learn
Operational Insights
Feedback integration yields prioritized remediation and roadmap adjustments.
Cycle 4. Consolidation & Late-Stage Optimization
In the last wet lab cycle, we consolidate the results of the previous cycles in a pipeline allowing to engineer and screen the activity of biocontrol strains against Phytophthora.
Engineering Iterations
Design
Final Design Hardening
Stabilizing design artifacts, freezing critical interfaces, and documenting invariants.
- Interface freeze checklist
- Failure mode review
- Risk retirements
- Traceability matrix closure
Design activity concentrates on eliminating ambiguity, tightening specifications and ensuring downstream build/test loops remain deterministic.
Build
Stability-Focused Assembly
Refined construction emphasizing reproducibility and quality lock.
Parallel builds benchmark variance; deviations feed corrective micro-adjustments and reagent qualification.
Test
Extended Reliability Trials
Burn-in style stress, extended shelf simulations and accelerated aging analyses.
Test focus: drift detection, degradation kinetics, performance envelope pinch points under compounded stress factors.
Learn
Refinement Synthesis
Aggregating late-cycle insights to finalize deployment dossier and improvement backlog.
Learning outputs: validated operational limits, prioritized improvement deltas, confirmed reproducibility thresholds.
Cycle 5. Agent-Based Modelling (ABM)
Use the iteration buttons to explore all three iterations of this cycle.
Engineering Iterations
Design
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Build
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Test
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Learn
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