iGEM 2025 — Team kuleuven

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## Table of Contents

* [Abstract](#abstract)
* [Summary](#summary)
* [References](#references)

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Lab / Wet lab / Noteboook /

## Notebook

Project 1

Project 2

Project 3

Project 4.

Project 5.

July

August

September

Assemblies

Assay

Assemblies

Assay

Iteration 1

Iteration 2

Assay

Assembly

## Project 1. Construction of the new parts in the SubtiToolKit (STK) syntax

#### Goal:

Thanks to the kind support of prof. Caro-Astorga, we received access to 96 different DNA parts functionable in B. subtilis. To facilitate the assembly of the full transcriptional units (TUs), we needed to produce our parts fused with appropriate overhangs, allowing the creation of DNA ends compatible with STK upon digestion with respective type IIS restriction enzymes.

#### Graphic abstract

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#### Results

We ordered the sequences of the [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
fused with respective overhangs, from IDT or Twist. Our first step was the resuspension of the Parts according to the producer’s guidelines. Next, the parts were either cloned directly to the entry vector pSTK-0-GFP through the Golden Gate assembly reaction with BbsI enzyme or first amplified with Q5 polymerase and cloned in the same reaction. White colonies (without the dropout GFP cassette) were first verified using colony PCR. Next, plasmids from the positive colonies were isolated and prepared for sequencing.
In the end, we constructed level 0 plasmids containing codon-optimized sequences of: phoA, AMPs, SUMO and thioredoxin.

#### Benchling export links:

## Project 2. Benchmark growth experiments

#### Goal:

The primary goal of these experiments is to systematically evaluate
different agar media to identify the most suitable conditions for the
growth of *Bacillus subtilis* and *Phytophthora capsici*  and *P. palmivora*.
This step is essential for our downstream competition assays, where our
transformed *B. subtilis* will be tested for its ability to inhibit
*Phytophthora* growth. We essentially want to test microbial growth as close
as possible to the competition assay conditions.
Additionally, we performed a calibration experiment to establish a correlation between optical density (OD₆₀₀) measurements and the actual colony forming unit (CFU) counts of *B. subtilis*. This calibration allows us to translate OD readings into reliable estimates of bacterial concentration for use in later experiments.

#### Graphic abstract

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#### Results

We repeated the assay thrice, each time including 4 wells with a
particular organism incubated on a particular media. Then, we used the
analysis tool of the Reshape Biotech Imaging Device to measure microbial
growth over time. Based on the results, we chose PSA as a medium for the
competition assays.

#### Benchling export links:

## Project 3. Set up of a secretion tag reporter assay in *B. subtilis*

#### Goal:

The goal of this assay was to engineer and test the constructs, in which
the production of alkaline phosphatase fused with different secretion tags
is induced by xylose - analogous to the AMP-containing constructs.

#### Graphic abstract

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#### Results

For the pilot assay, we assembled constructs with phoA fused with two
different secretion tags: SacB and Apr, or a Cx linker. We used two
different RBS sequences in the assembly - a strong RBS used frequently
with the popular Phyperspank promoter, or an RBS equipped with the
sequence stopping the translation in *E. coli* to prevent potential
toxicity. In the end, we obtained fully correct sequences of the
constructs with Apr secretion tag, Cx linker, and SacB with the RBS
preventing translation in *E. coli*.
In the assay, we employed both positive (a purified alkaline phosphatase
enzyme from Promega) and negative controls (samples from wild type *B.
subtilis* 168). We tested supernatant and cell lysate samples from all *B.
subtilis*  strains. In the end, we saw clear, reproducible signal from the
positive control, little background signal from the negative controls, and
clear signal from the Apr construct assembled with the RBS blocking
translation in *E. coli*.

#### Benchling export links:

## Project 4. Engineering of *B. subtilis* strains expressing AMPs

#### Goal:

The aim of the main project of the wet lab team was the construction of
plasmids allowing for the production and secretion of antimicrobial
peptides by *B.
subtilis* , using parts developed by Caro-Astorga et al.
2025, as well as basic parts incorporated into STK syntax by our team in
the previous weeks.

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#### Results

After many rounds of the optimization of the GGA cloning, we obtained the
following constructs:

* Plasmids encoding for the vicilin-like antimicrobial peptide 2-2 with
  both **Cx** linker and **SacB** secretion tag
* Plasmid encoding for MIAMP 2-2 with **Cx** linker
* Plasmids encoding for cecropin with both **Cx** linker and
  **SacB** secretion tag

*B. subtilis* strains transformed with the above-mentioned plasmids
were tested for their ability to inhibit *Phytophthora* growth in the
dual culture assay established in the previous weeks. While we did not see
a significant inhibitory effect, our [experimental pipeline](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) can be re-used
in the future for the development of reliable assays measuring fungal or
oomycete growth inhibition.

#### Benchling export links:

## Project 5. Purification of SUMO-AMP fusions

#### Goal:

The versatility of our cloning approach allows us to seamlessly switch
between cloning of the secretion tag-AMP fusions to purification tag-AMP
fusions, just by exchanging one DNA part in the Golden Gate assembly. The
goal of this project was to assess the efficiency of the cloning and
purification of SUMO-tagged AMPs produced by B. subtilis.

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#### Results

After a few rounds of the optimization of the GGA cloning, we obtained a
plasmid allowing for the expression of the SUMO-AMP03 (cecropin) fusion
(described in the entry related to “Construction of E.coli - B. subtilis
shuttle vectors for the expression of AMPs”). We transformed B. subtilis
with the construct but struggled to obtain the sufficient growth of the
engineered strain.

#### Benchling export links:

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Engineering innovative solutions for sustainable biocontrol - Designing robust systems for detection, production, and delivery of antimicrobial peptides.
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## Table of Contents

* [Summary](#custom-hero)
* [Cycle 1: SubtiToolKit Preparation](#engineering-cycle)
* [Cycle 2: Secretion Assay Setup](#engineering-cycle-2)
* [Cycle 3: Dual Culture Assay](#engineering-cycle-3)
* [Cycle 4: Consolidation & Optimization](#engineering-cycle-4)
* [Cycle 5: Agent-Based Modeling](#engineering-cycle-5)

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## Cycle 1. Preparation of the new parts in the SubtiToolKit syntax

Design

Build

Test

Learn

Cycle 1 focuses on the implementation of new basic parts compatible with the SubtiToolKit syntax.

### Engineering Iterations

Iteration 1
Iteration 2

❮
❯

### 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.

![Design Phase - System Architecture](https://static.igem.wiki/teams/5691/engineering/fig-1.webp)

**Figure 1:** SubtiToolKit (STK) assembly workflow showing the systematic approach from Level 0 entry vectors to Level 1 functional constructs through Golden Gate assembly.

STK relies on the assembly of parts pre-cloned to Level 0 (“entry”) vectors derived from pSTK-0-GFP vector. After digestion with BsaI and subsequent ligation, Level 0 plasmids can be assembled into Level 1 plasmids (Fig. 1). To ensure the high efficiency of the final assembly, the nucleotide sequences of AMPs should be cloned into level 0 vectors as well.

The aim of the first engineering cycle was:

1. To obtain the nucleotide sequences of our parts and,
2. To assemble Level 0 plasmids.

We decided to codon-optimize the sequences for the optimal expression in *B. subtilis* – an approach proven to enhance the production rates of AMPs [2], [3]. After long discussions, we decided to add Cy overhangs to our parts – allowing us to fuse AMP with one protein on N-terminal end and one protein/tag on C-terminal end.

### 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

![Test Phase - Validation](https://static.igem.wiki/teams/5691/project/description/low-angle-shot-cocoa-tree-with-blooming-cocoa-beans-it.webp)

**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*

Design

Build

Test

Learn

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

Iteration 1

❮
❯

### Design

#### Advanced System Architecture & Optimization

Enhanced system architecture incorporating lessons learned from Cycle 1, with focus on scalability, efficiency, and production readiness.

![Design Phase - Advanced Architecture](https://static.igem.wiki/teams/5691/project/description/image-1.webp)

**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

![Build Phase - Optimized Construction](https://static.igem.wiki/teams/5691/project/description/construct-design.webp)

**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

![Test Phase - Enhanced Validation](https://static.igem.wiki/teams/5691/project/description/low-angle-shot-cocoa-tree-with-blooming-cocoa-beans-it.webp)

**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

![Learn Phase - Optimization Analysis](../../static/images/cacao-approach.webp)

**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

Design

Build

Test

Learn

In this cycle, we optimize the dual-culture assay conditions by measuring the microbial growth in different media and temperatures .

### Engineering Iterations

Iteration 1
Iteration 2

❮
❯

### 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

Design

Build

Test

Learn

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

Iteration 1
Iteration 2
Iteration 3

❮
❯

### 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)

Design

Build

Test

Learn

Use the iteration buttons to explore all three iterations of this cycle.

### Engineering Iterations

Iteration 1
Iteration 2
Iteration 3

❮
❯

### Design

#### (Add Design Title)

Placeholder overview for Cycle 5 Design phase.

Detailed descriptive content for Cycle 5 Design phase goes here.

### Build

#### (Add Build Title)

Placeholder overview for Cycle 5 Build phase.

Detailed descriptive content for Cycle 5 Build phase goes here.

### Test

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Placeholder overview for Cycle 5 Test phase.

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### Learn

#### (Add Learn Title)

Placeholder overview for Cycle 5 Learn phase.

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## References

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26. A. P. Sikkema, S. K. Tabatabaei, Y.-J. Lee, S. Lund, and G. J. S. Lohman, “High-Complexity One-Pot Golden Gate Assembly,” *Curr. Protoc.*, vol. 3, no. 9, p. e882, 2023, doi: 10.1002/cpz1.882.
27. A. M. Rodd, A. Jawad, and F. E. Nano, “Bacillus subtilis small replicative plasmid collection incorporating five distinct origins of replication and compatible with Golden Gate DNA assembly,” *FEMS Microbiol. Lett.*, vol. 370, p. fnad039, Jan. 2023, doi: 10.1093/femsle/fnad039.
28. D. Cai, Y. Rao, Y. Zhan, Q. Wang, and S. Chen, “Engineering Bacillus for efficient production of heterologous protein: current progress, challenge and prospect,” *J. Appl. Microbiol.*, vol. 126, no. 6, pp. 1632–1642, Jun. 2019, doi: 10.1111/jam.14192.
29. X. C. Wu, W. Lee, L. Tran, and S. L. Wong, “Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases,” *J. Bacteriol.*, vol. 173, no. 16, pp. 4952–4958, Aug. 1991, doi: 10.1128/jb.173.16.4952-4958.1991.
30. T. T. Nguyen, T. D. Quyen, and H. T. Le, “Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800,” *Microb. Cell Factories*, vol. 12, no. 1, p. 79, Sep. 2013, doi: 10.1186/1475-2859-12-79.
31. R. C. Judd, “Electrophoresis of Peptides,” in *Basic Protein and Peptide Protocols*, J. M. Walker, Ed., Totowa, NJ: Humana Press, 1994, pp. 49–57. doi: 10.1385/0-89603-268-X:49.
32. P. F. Popp, M. Dotzler, J. Radeck, J. Bartels, and T. Mascher, “The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis,” *Sci. Rep.*, vol. 7, no. 1, p. 15058, Nov. 2017, doi: 10.1038/s41598-017-15107-z.
33. J. Du et al., “Elicitin recognition confers enhanced resistance to Phytophthora infestans in potato,” *Nat. Plants*, vol. 1, no. 4, p. 15034, Mar. 2015, doi: 10.1038/nplants.2015.34.
34. M. Haghpanah, A. Namdari, M. K. Kaleji, A. Nikbakht-dehkordi, A. Arzani, and F. Araniti, “Interplay Between ROS and Hormones in Plant Defense Against Pathogens,” *Plants*, vol. 14, no. 9, p. 1297, Apr. 2025, doi: 10.3390/plants14091297.

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![Close sidebar](data:image/svg+xml... "Close side bar")

## Table of Contents

* [Molecular Biology](#molecular-biology)
* [High-fidelity PCR](#high-fidelity)
* [Restriction Digestion](#restriction-digestion)
* [Golden Gate Assembly (Type 0)](#golden-gate-assembly)
* [Golden Gate Assembly (Type 1)](#type-1-assembly)
* [Competent Cells Preparation - E. coli](#competent-cells-preparation)
* [Transformation - E. coli](#transformation)
* [Competent Cells Preparation - B. subtilis](#competent-cells-b-subtilis)
* [Transformation - B. subtilis](#transformation-b-subtilis)
* [Media Protocols](#media-protocols)
* [Assay Protocols](#assay-protocols)
* [Competition Assay](#competition-assay)
* [Growth Assay](#growth-assay)
* [Alkaline Phosphatase Assay](#alkaline-phosphatase)
* [OD₆₀₀ to CFU Calibration](#od-cfu-calibration)
* [References](#references)

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## Molecular Biology

### Colony PCR using GoTaq® G2 Master Mix from Promega

The aim of this experiment was to validate the occurrence of a specific DNA sequence in bacteria grown on a solid medium. It is commonly used for selecting clones bearing the desired construct after transformation of competent bacterial cells with ligation mixtures. For more detailed guidelines on primer design and technical setup, see [1].

This protocol is based on the guidelines of the manufacturer (Promega), and on a general protocol from Prof. Pinheiro's Lab.

Read more

- GoTaq® G2 Master Mix (catalog nr M7822)
- Nuclease-free water
- Primer pair: forward and reverse, 10µM each
Equipment

* Thermocycler
* Thermoblock
* Table-top centrifuge

#### Procedure

1. Lysis of bacteria
   1. Fill a 1.5mL tube with 10 µL of nuclease-free water.
   2. Pick the colonies/scrape some material from the plate/tubes with glycerol stocks:
      1. Colonies: After ligation, transformation, and growth of the bacteria, pick 3-8 colonies/variants.
      2. Glycerol stock: Keeping the plate/tube on dry ice, scrape some of the ice and transfer to the PCR tube.
   3. Vigorously wash the tip or toothpick in the PCR tube.
      1. For the colonies: Without contaminating the tip or toothpick, streak it in a LB Agar plate with the corresponding antibiotic.
   4. Lyse the cells for 10 minutes at 95°C, using the thermoblock.
   5. Spin down the debris at 2000-g for 5 minutes.
   6. Use the supernatant as a matrix in your reaction.
2. Reaction mixture (total volume 25µl)

   | Reagent | Volume [µl] |
   | --- | --- |
   | GoTaq® G2 Master Mix | 12.5 |
   | 10 µM Forward Primer | 0.5 |
   | 10 µM Reverse Primer | 0.5 |
   | Supernatant of the lysed cells | 2.5 |
   | Nuclease-free water | 9.0 |

   #### Note: The reaction volume can be successfully halved to 12.5µl.
3. PCR reaction steps:

   |  | Temperature | Time |
   | --- | --- | --- |
   | Initial Denaturation | 95°C | 30 seconds |
   | 25–30 Cycles | 95°C | 30 seconds |
   | X | 30 seconds |
   | 72°C | Y |
   | Final Extension | 72°C | 5 minutes |
   | Hold | 16°C | forever |

   **X** – annealing temperature; it is recommended to start with temperature 5°C below the lowest melting temperature of the chosen primers.
   **Y** – elongation time, depends on the size of the expected product and DNA synthesis speed of a chosen polymerase (here, 1 kb/min).

## High-fidelity PCR using Q5® High-Fidelity 2X Master Mix from New England Biolabs

The aim of this experiment was to amplify a DNA fragment with high fidelity. According to the provider’s website, Q5 polymerase used in this mix offers fidelity over 280x greater than Taq polymerase, significantly lowering the error rate and thus, the introduction of mutations.

In our project, we used Q5® High-Fidelity polymerase to amplify and add overhangs to our gBlocks. This increased the variety of parts that we could use in the assembly while ensuring an extremely low rate of mutations acquired during amplification.

Read more

#### Materials

* GoTaq® G2 Master Mix (catalog nr M7822)
* Nuclease-free water
* Primer pair: forward and reverse, 10µM each

Equipment

* Thermocycler
* Thermoblock
* Table-top centrifuge

#### Procedure

1. General reaction setup

   | Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
   | --- | --- | --- | --- |
   | Q5 High-Fidelity 2X Master Mix | 12.5 µl | 25 µl | 1X |
   | 10 µM Forward Primer | 1.25 µl | 2.5 µl | 0.5 µM |
   | 10 µM Reverse Primer | 1.25 µl | 2.5 µl | 0.5 µM |
   | Template DNA | Variable\* | Variable\* | < 1,000 ng |
   | Nuclease-Free Water | to 25 µl | to 50 µl |  |

   In general, we added 5ng of DNA to the 50 µl reaction mixture
2. PCR reaction steps:

   | STEP | TEMPERATURE | TIME |
   | --- | --- | --- |
   | Initial Denaturation | 98°C | 30 seconds |
   | 25–35 Cycles | 98°C | 5–10 seconds |
   | X | 10–30 seconds |
   | 72°C | Y |
   | Final Extension | 72°C | 2 minutes |
   | Hold | 4–10°C |  |

**X** – annealing temperature of the primers. It is recommended to try the one recommended by the manufacturer (<https://tmcalculator.neb.com/#!/main>)
**Y** – elongation time, depends on the size of the expected product and DNA synthesis speed of a chosen polymerase (here, 1kb/20–30s)

## Restriction Digestion

Restriction digestion cleaves DNA in sites determined by short, palindromic sequence, recognized by a specific restriction enzyme. This technique is most commonly used in cloning (followed by ligation) or as a method of linearizing a plasmid to verify its size using gel electrophoresis.

In our project, we used type IIS restriction enzymes in Golden Gate Assembly reactions, or a type II enzyme to verify the size of our assembled plasmids (this protocol).

Read more

#### Materials

* rCutSmart™ Buffer (New England Biolabs)
* Chosen restriction enzyme (New England Biolabs)
* Nuclease-free water

#### Equipment

* Thermoblock

#### Procedure

1. Prepare the reaction mix. Gently mix the components by pipetting up and down, spin down shortly.

   | Component | Amount for 25 µl reaction mix [µl] | Final concentration/amount |
   | --- | --- | --- |
   | rCutSmart™ Buffer | 2.5 | 1x |
   | Restriction enzyme | 0.5\* | Variable\* |
   | DNA sample | Variable\*\* | Up to 1 ng |
   | Nuclease-free water | Up to 25 µl |  |

   \* Enzyme-dependent. Usually, we added 10U of the enzyme of choice to the reaction mixture
   \*\* Usually, we digested around 500 ng plasmid DNA in the 25 µl reaction
2. Incubate at the temperature suggested by the provider.
   1. For NEB enzymes:
      <https://www.neb.com/en/tools-and-resources/usage-guidelines/nebbuffer-performance-chart-with-restriction-enzymes?srslitid=AfmBOoryXgkfwwQdjCP_pmRzCALmY04Ce2NNb3c_8u0RcKmZCCoXukLS>
   2. **Note:** In our experiments we used Time-Saver™ Qualified Restriction Enzymes, which allowed us to reduce the restriction digest time to 15 minutes.
3. Inactivate at the temperature suggested by the provider or add a gel loading dye to immediately run the sample on the gel.

## Golden Gate Assembly – type 0 vectors (BbsI)

The aim of this protocol was to assemble level 0 vectors with new parts compatible with the SubtiToolKIt. Here, the entry vector (STK001\_pSTK-0-sfGFP) and a part of choice (gBlock) are cleaved by BbsI and ligated with T4 ligase.

The protocol is based on the one from the SubtiToolKit and additional guidelines from our PI. Initially, we noticed a low efficiency of cloning in preparation of our alkaline phosphatase (phoA) plasmids as described in the  [notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#hredd) . After consulting with our PI, we decided to separate the steps of digestion and ligation. To ensure that the ligase is not deactivated during the incubation step, this protocol involves a two-step process whereby the plasmid and insert are digested with the restriction enzyme and then ligated in a second step requiring the addition of the ligase. We tested different incubation time conditions.  [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#hredd) .
Based on the results, we chose 15 minutes incubation with BbsI and 15 minutes incubation with T4 as the incubation times.

Read more

#### Equipment:

- Thermoblock
- Table-top centrifuge

#### Materials

* BbsI-HF® (R3539S)
* rCutSmart™ buffer (B6004S)
* T4 ligase (M0202L)
* T4 ligase reaction buffer (B0202S)
* Nuclease-free water

#### Procedure

##### Restriction

1. Prepare the reaction mix (total volume 20 µl). To calculate the needed volume of vector and insert, refer to the https://nebiocalculator.neb.com/#!/dsdnaamt calculator.

   | Component | Volume needed [µl] | Final concentration/amount |
   | --- | --- | --- |
   | Vector | variable | 50 fmol |
   | Insert | variable | 100 fmol |
   | RCutSmart buffer 10X | 2 | 1X |
   | BbsI | 1 |  |
   | Nuclease-free water | Up to 20 µl |  |
2. Incubate 15 minutes at 37°C

##### Ligation

1. Prepare the reaction mix with a total volume of 10 µl. Add the ligation mix to the restriction mix.

   | Component | Volume needed [µl] |
   | --- | --- |
   | T4 Ligase buffer 10x | 3 |
   | T4 Ligase | 0.5 |
   | Water | 6.5 |
2. Incubate for 15 minutes at room temperature
3. Transform *E. coli* with the prepared ligation mixture or store temporarily at 4°C

## Golden Gate Assembly – type 1 vectors (BsaI)

Type IIS enzymes enable effortless assembly of DNA constructs due to cutting a few nucleotides outside of the recognition sequence. Golden Gate Assembly (GGA) is a one-pot, one-reaction cloning method, most frequently involving Type IIS enzymes like BsaI or SapI. The main advantage of GGA is the possibility of designing compatible ends allowing to assemble many DNA parts in the same reaction.

The SubtiToolKit relies on GGA to clone the parts into full transcriptional units (TUs). Each TU consists of a promoter, ribosome binding site (RBS), coding sequence (CDS; can be split into three parts – Cx, Cy, Cz – to accommodate N- and C-terminal tags) and a terminator. The resulting plasmid is a – shuttle vector between *E. coli* and *Bacillus subtilis*. The utilization of a specific syntax enables efficient cloning of the parts in the correct order.

Described below is the final protocol used by our team to assemble the parts (our troubleshooting steps were further described in the engineering cycle 4 and the  [notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D#hredd) ).

Read more

#### Equipment:

* Thermoblock
* Table-top centrifuge

#### Procedure

##### Restriction

1. Prepare the reaction mixture

   | Component | Volume needed [µl] | Comments |
   | --- | --- | --- |
   | DNA parts | variable | 50 fmol/each plasmid |
   | RCutSmart buffer | 2 |  |
   | BsaI | 1 |  |
   | Nuclease-free water | Up to 10 µl |  |
2. Incubate for 1 hour in 37°C

##### Ligation

1. Prepare the ligation mixture

   | Component | Volume needed [µl] | Final concentration/amount |
   | --- | --- | --- |
   | Restriction mixture | 10 |  |
   | T4 ligase (200U/mL) | 0.5 | 50U |
   | T4 ligase buffer 10X | 4 | 1X |
   | PEG3000 30% | 4 | 3% |
   | Nuclease-free water | Up to 40 µl |  |
2. Incubate for 5 minutes in 16°C
3. Incubate for 20 minutes in 60°C
4. Transform immediately or store in –20°C

## Chemo competent cells preparation – *E. coli* DH10b

This is a standard protocol, adapted by Prof. Pinheiro's lab. The prepared competent cells are stored in –80°C freezer and used in the transformation experiments described below.

Read more

#### Materials

* 100 mM CaCl2 solution
* 100 mM MgCl2 solution
* 85 mM CaCl2 with 15 % glycerol solution
* Fresh LB medium without antibiotics

#### Equipment

* Refrigerated centrifuge

#### Procedure

1. Grow overnight culture of desired *E.coli* strain
   1. Note: we inoculated bacteria from the glycerol stocks in 5 mL LB, cultured in 50 mL falcon tube
2. Add 1/100 of the overnight culture to fresh LB media
   1. Note: we added 2 mL of the o/n culture to ~200 mL fresh LB
3. Grow to OD600 = 0.4
4. Split the culture into falcon tubes, centrifuge at 4°C at 4000 g for 6 minutes
   1. Note: we split our culture into 4 falcons, 50 mL culture each
5. Discard the supernatant and resuspend pellets in cold 100 mM MgCl2
   1. Note: we resuspended the pellets in 15 mL 100 mM MgCl2/falcon
   2. Avoid pipetting up and down during resuspension – instead, gently tap the falcon on the palm of your hand
6. Centrifuge at 4°C at 3000 g for 6 minutes
7. Discard the supernatant and resuspend pellets in cold 100 mM CaCl2
   1. Note: we resuspended the pellets in 2.5 mL 100 mM CaCl2/falcon
   2. Pipet gently up and down
8. Combine the content of the falcon tubes in one tube
9. Centrifuge at 4°C at 2000 g for 15 minutes
10. Discard the supernatant and resuspend the pellets in 2 mL 85 mM CaCl2, 15% glycerol
11. Aliquot 50 µl/tube and place immediately on dry ice
12. Store at –80°C

## Transformation– *E. coli* DH10b

This protocol is based on the classic protocol for *E. coli* transformation, adapted by the Pinheiro lab.

Read more

#### Materials

* Fresh LB
* Chemocompetent cells
* DNA

Equipment

* Thermoblock
* 37°C incubator

#### Procedure

1. Add 5 µl of the cloning reaction mixture to competent cells, on ice
2. Incubate on ice for 30 minutes
3. Heat shock at 42°C for 30 seconds
4. Immediately transfer the cells to ice and incubate for 5 minutes
5. Add 450 µl of pre-warmed LB medium
6. Incubate for 60 minutes in 37°C with shaking
   1. Note: in our case, incubation time ranged from 45 to 75 minutes, and the shaking speed was 180 rpm
7. Plate the cells on the pre-warmed plates
   1. Note: when plating “high” and “low” concentration of cells, we first plated 50 µl of the transformation mixture. Next, we centrifuged the tubes for 3 minutes at 3000 g, discarded the supernatant “in a swing”, resuspended the pellet in the media remaining at the bottom of the tube (around 50 µl) and plated the rest of the mixture
   2. We spread the bacterial cultures on the plates using glass beds. First, we added 5–10 sterile beds onto each plate, then we added the desired volume of the culture onto 1 to 2 beds, closed the lid and shook gently back and forth until the liquid was uniformly spread over the whole plate
8. Incubate at 37°C

## Chemo competent cells preparation – *B. subtilis* 168

Some isolates of *B. subtilis* can uptake foreign DNA upon reaching stationary growth phase. This state of natural competence is determined by quorum sensing and nutrient limitation [2]. Importantly, not every *B. subtilis* isolate can be transformed by using the method described below [3]. The protocol below was validated for *B. subtilis* DB104 [4] and *B. subtilis* 168 [5]. This protocol is based on the natural competence protocols described by Caro-Astorga et al. [5] and Falkenberg et al [6].

Read more

#### Materials

* 10x SM1 medium stock
  + 2 g ammonium sulphate
  + 14 g dipotassium dihydrogen phosphate
  + 6 g potassium dihydrogen phosphate
  + 0.7 g sodium citrate
  + 0.2 g magnesium sulphate heptahydrate
  + 2 g yeast extract
  + 0.25 g casamino acids
  + MiliQ until 100 ml
* 1x SM2 medium stock
  + 2 g ammonium sulphate
  + 14 g dipotassium dihydrogen phosphate
  + 6 g potassium dihydrogen phosphate
  + 0.7 g sodium citrate
  + 0.8 g magnesium sulphate heptahydrate
  + 1 g yeast extract
  + 0.1 g casamino acids
  + MiliQ until 1 L
* 200 g/l glucose stock
* 1M CaCl2 stock
* 50% glycerol

#### Equipment

* Incubator 37°C
* -80°C freezer

#### Procedure

1. Grow *B. subtilis* overnight at 37°C in 1x SM1 medium supplemented with 5 g/L glucose.
   1. First dilute 10x stock with 9 mL sterile H2O. Add 25 µl of the glucose stock per mL of the medium.
   2. Note: we inoculated *B. subtilis* from a freshly streaked plate into 5 mL LB medium in 50 mL sterile falcon tube.
2. Measure the OD600 of the overnight culture and dilute it in 250 mL flask to reach OD600 = 0.5 in 10 mL of SM1 medium supplemented with 5 g/L glucose.
   1. Note: for example, if the OD600 of your culture is 1.0, you need to mix 5 mL of your culture with 5 mL of SM1 medium in 250 mL sterile flask.
3. Grow the culture at 37°C with shaking for 3 hours.
   1. Note: the original protocol mentions 250 rpm as the shaking speed. In our case, we used 180 rpm.
4. Add 10 mL of SM2 medium followed by 250 µL of 200 g/L glucose stock.
5. Add 45 µL of the 1M CaCl2 solution while swirling the flask.
6. Continue incubation at 37°C with shaking for 2 more hours.
7. Aliquot the cells into 500 µl portions in 2 mL sterile tubes. Add 250 µl of 50% glycerol to the aliquots, mix gently and store at –80°C.

## Transformation – *B. subtilis*

This protocol enables the transformation of the competent cells of *B. subtilis*. It is based on the natural competence protocols described by Caro-Astorga et al. [5] and Falkenberg et al [6].

Read More

#### Equipment:

* Incubator 37°C
* Thermoblock

#### Materials

* LB medium
* LB agar medium supplemented with desired antibiotic concentration
* An aliquot of the *B. subtilis* competent cells
* Plasmid

#### Procedure

1. Thaw the competent cells on ice.
2. Add 250 ng–1 pg of plasmid DNA to the competent cells.
3. Incubate in the thermoblock at 37°C for 30 minutes with 800 rpm shaking.
4. Add 300 µl of LB medium to each aliquot and incubate at 37°C with 800 rpm shaking for at least 2 hours.
5. Plate 200 µl of the recovery culture on LB plates
   1. Note: As with *E. coli* transformation, it is possible to increase the colony count by centrifugation of the samples at 6000g for 2 minutes
6. Incubate overnight at 37°C.

## Media protocols

Read More

1. ### **V8 Juice Agar (Clarified)**

   #### Purpose

   Plant-juice–based medium for certain fungi/oomycetes.

   #### Materials

   1. 120 mL V8 juice (unfiltered)
   2. 5 g CaCO3
   3. 12 g Agar
   4. 480 mL tap water

   #### Protocol

   **20% clarified V8-medium**
   1. Add 5 g of CaCO3 to 340 mL V8-juice in a beaker containing a stir bar.
   2. Stir for at least 15 minutes.
   3. Aliquot into 50 mL Falcon tubes, making sure the tubes will be balanced in centrifuge – either by weighing or using gradations of tubes.
   4. Spin tubes in a centrifuge at 10,000 rpm for 15 minutes.
   5. Transfer clarified V8-medium to 50 mL aliquots for storage at –20 °C.
   **Making of 20% CV8-medium**
   1. Add 60 mL of the clarified V8 juice to 240 mL distilled water.
   2. 9 g agar is used for 300 mL liquid.
   3. Mix and autoclave (15 min at 121 °C).

2. ### 2. Potato Dextrose Agar (PDA)

   General-purpose fungal medium; typically prepared according to the manufacturer’s label.

   #### Materials

   1. 39g of Merck PDA powder

   #### Protocol

   1. Weigh the Merck PDA powder
   2. Add the powder to ~900mL of distilled water in a flask or beaker
   3. Stir and heat gently until fully dissolved, make sure no clumps remain
   4. Bring the total volume to 1L by adding distilled water
   5. Autoclave at 121°C for 15min
3. ### 3. Pea Sucrose Agar (PSA)

   Legume extract plus sucrose medium

   #### Materials

   1. 96g frozen garden peas (no salt!!)
   2. 3g sucrose
   3. 9g agar

   #### Protocol

   **Pea extract**
   1. Blend the peas with ~400mL–300mL distilled water until very smooth.
   2. Boil gently for 15–20 min.
   3. Filter through cheesecloth.**Combine**
   1. Combine pea filtrate, 3g sucrose, 9g agar.
   2. Make up the volume to 600mL using distilled water.**Sterilize**
   1. Autoclave 121°C for 15 min.
4. ### 4. Carrot Agar (CA)

   Root-vegetable–based medium often used for culturing and sporulation studies of certain plant-associated fungi/oomycetes.

   #### Materials

   1. 12g potatoes (diced)
   2. 12g carrots (diced)
   3. 9g agar
   4. 600mL distilled water

   #### Protocol

   **Prepare extract**
   1. Simmer potatoes and carrots in ~300–400mL distilled water for 30 min
   2. Mash during or after boiling
   3. Strain through cheesecloth to remove solids**Combine**
   1. Add filtrate to a flask, add 9g agar and bring to 600mL volume with distilled water**Sterilization**
   1. Autoclave at 121°C for 15 min

## Assay protocols

### *Phytophthora* Storage & Revival

Read More

#### Materials

* Pipette and pipette tips
* Round petri plates with media of choice (for example PDA)
* *P. palmivora* and *P. capsici* stock
* Cryovials with screw caps
* Cork borer
* Sterilized tap water (121°C for 20 min)

#### Propagation protocol

1. Transfer a single mycelium plug from the *phytophthora* stock available, taken with a cork borer, to the centre of round petri plates containing your agar of choice.
2. Let it grow for 4–10 days.
3. These plates can now be used for upcoming experiments.

#### Long-term storage

1. If you want to have some long-term storage you can fill a cryovial with 1 mL sterilized tap water and put max. 5 mycelium plugs in the cryovial.
2. Close the vial with a screw cap.
3. These can be kept at room temperature (18–25°C) in the shade for max. 6 months.

## Competition assay

Read More

#### Materials:

* Round Petri dishes
* Pipette and pipette tips
* PSA plates containing Erythromycin and xylose 1%
* 6-14 days old PDA plates of *P. palmivora* and *P. capsici*
* Liquid bacterial cultures of choice (at least 1 day growth)

#### Before starting

1. Transfer a mycelium plug with a sterile cork borer and forceps from an actively growing mycelium edge of *P. palmivora* and *P. capsici* to a new plate of PDA agar. Place the mycelium plug in the centre of the Petri dish. **Do this 1 time for each.**
2. Incubate at least 4 days at 27°C.
3. Start an overnight culture of desired bacteria, **1 day before the start of the experiment.**

#### Day of experiment

1. Measure OD of overnight bacterial culture using the Spectrophotometer, desired OD is 0.3
   1. Note: make necessary dilutions if OD is higher the day of the experiments.
2. Transfer a mycelium plug with a sterile cork borer and forceps from an actively growing mycelium edge of *P. palmivora* and *P. capsici* to the centre of a round petri dish containing PSA agar with Erythromycin and 1% xylose.
3. Then add 20µl (via reverse pipetting) of the bacterial suspension, this is spotted three times, 1.5 cm away from the border of the Petri dish, making sure the angle between each bacterium is ≈120°.
   1. Note: Very important after adding bacterial suspension do not tilt plates! If possible, leave plate to dry 10–15 min before moving out of the hood.
4. Seal the plates with parafilm.
5. Incubate 25–27°C for at least 5 days in an incubator.
6. Analyse plate by taking pictures every 2 hours and tracking growth using ImageJ.

## Protocol: Growth Assay of *Phytophthora* spp. and *B. subtilis* on Xylose-Supplemented Agar

Read More

#### Materials:

* **Phytophthora* capsici* and **Phytophthora* palmivora* stock cultures (PDA plates)
* *B. subtilis* glycerol stock
* LB Broth
* Potato Dextrose Agar (PDA)
* Xylose (3 g per 20 mL water)
* Distilled water
* 0.22 µm filter
* SBS 6-well plates
* Cork borer (5 mm)
* Sterile pipette tips
* Sterile spreader beads

### Preparation:

**Four days prior to experiment:**

1. Plate mycelium plugs of *P. capsici* and *P. palmivora* on PDA plates.
2. Incubate at appropriate temperature to serve as stock cultures for benchmarking growth.

**One day prior to experiment:**

3. Inoculate *B. subtilis* from glycerol stock into LB broth.
4. Incubate overnight at 37°C with shaking to reach OD600 ≈ 0.4.

**Agar Preparation**

5. Prepare media according to protocol.
6. Dissolve 3 g xylose in 20 mL distilled water.
7. Filter-sterilize xylose solution using a 0.22 µm filter.
8. Allow PDA to cool to a pourable temperature (~50°C), then add xylose solution and mix gently.
   1. Note: Ensure agar is not too hot before adding xylose to avoid sugar degradation.
9. Pour media into SBS 6-well plates, filling approximately half of each well.
10. Allow agar to fully solidify.

**Inoculation**

**Phytophthora* spp.:*

1. Using a sterile 5 mm cork borer, obtain mycelium plugs from stock cultures.
2. Place one plug in the centre of four of the agar wells.

*B. subtilis:*

1. Bring overnight culture to OD600 ≈ 0.4
2. Dilute overnight culture to 10–5.
3. Spot 50 µL of diluted culture onto sterile spreader beads.
4. Spread bacteria evenly over the surface of the remaining agar wells.
5. Incubate the plate at the desired temperature and measure growth by taking pictures every 30 minutes and tracking growth using ImageJ.

## Alkaline phosphatase reporter assay

Alkaline phosphatase (AP) is a commonly used reporter enzyme in molecular biology. It catalyzes the removal of phosphate groups from various molecules, including nucleotides and proteins. The activity of AP can be easily measured using colorimetric or fluorometric substrates, making it a valuable tool for assessing gene expression and promoter activity in bacterial systems. In this protocol, we describe the steps to perform an alkaline phosphatase reporter assay in *B. subtilis*.This assay enables the measurement of the activity of alkaline phosphatase (PhoA) produced and secreted by *B. subtilis*. In brief, bacterial samples are incubated with para-Nitrophenyl phosphate (pNPP). The enzymatic activity is reported by measuring the breakdown of pNPP to yellow para-Nitrophenyl (pNP) (OD410 measurement).

Read More

#### Equipment:

* Plate reader
* Centrifuge
* 37°C incubator

#### Materials:

* LB
* 10% xylose solution (filter sterilized)
* Lysis solution
  + SDS 0.01% (prepare 10% stock solution)
  + 1 mg/ml hen egg lysozyme (store in fridge)
* Substrate buffer (store in the fridge)
  + 2 mL of 50 mM Tris-HCl, pH=8
  + 2.5 mL of 0.5% pNPP
* 96-well microplates
  + We used F-bottom Greiner microplates

#### Procedure:

**Preparation of cell lysate and culture supernatant samples**

1. Prepare overnight cultures of the tested *B. subtilis* strains.
2. In the morning, dilute the cultures in fresh LB medium to OD600 = 0.1
   1. In the cuvette, mix 250 µl of culture and 750 µl of LB
   2. To prevent the contamination of the lab with *B. subtilis* spores, seal the cuvettes with parafilm since they contain *B. subtilis* before removing them from the hood
   3. Total volume of the culture should be 5 mL. Prepare 3 replicates/construct
3. Incubate in 37°C on the shaker until the cultures reach OD600 = 1 (approximately)
   1. Check the OD600 after 1:30h. Afterwards, if needed, check again in half hour increments
4. Add xylose to 1% final concentration
   1. From a 10% stock of xylose, add 500μl to 5mL culture
5. Incubate at 37C on shaker for 3 hours.
6. Centrifuge at 5000g for 10 minutes at room temperature
   1. It is also possible to transfer 1 mL of the culture sample to eppendorf tubes and spin them in a tabletop centrifuge for 5 minutes at 8000g
   2. Keep both supernatant and pellet samples. Transfer supernatant to a new falcon and set aside.
7. Resuspend pellets in 5mL of Lysis solution
8. Centrifuge the lysed pellet samples for 15 minutes at 5000g at room temperature; keep the supernatant

#### Preparation of the reaction mixture and measurements

1. To each well in 96-well plate add:
   1. 100uL of the Substrate Buffer
   2. 100uL of the bacterial sample/standard
2. Use a plate reader device to measure the PhoA activity over time
   1. Our settings: OD410 measurement every 30s for 30 min; incubation in room temperature with shaking
3. Data analysis

We analysed our results by preparing two types of graphs:

* Tracking the accumulation of the coloured product (OD410) over time
* End-point OD410 measurements, allowing to compare the enzymatic activity in supernatant and cell lysate samples

## Calibration of OD₆₀₀ to CFU for *B. subtilis* 168

To establish a correlation between optical density (OD₆₀₀) readings and viable cell counts (CFU/mL) for *B. subtilis* 168, enabling accurate estimation of bacterial concentration from OD measurements in future experiments.

Read More

### Materials

* *B. subtilis* 168 overnight culture (grown in LB broth)
* LB broth (sterile, autoclaved)
* 500 mL Erlenmeyer flask with grooves (for aeration)
* Sterile 1.5 mL microcentrifuge tubes
* Sterile cuvettes
* Spectrophotometer for OD600 measurement
* Pipettes and sterile tips
* Sterile 15 mL conical tubes
* Agar plates (LB agar)
* Dilution buffer or sterile LB for dilutions
* Sterile spreaders

#### Procedure:

**Overnight Culture Preparation**

1. Inoculate *B. subtilis* 168 into 5 mL of LB broth.
2. Incubate overnight at 37°C with shaking (200 rpm).
3. Estimating Initial Culture Concentration
4. Dilute the overnight culture 1:10 in LB broth.
   1. Example: 100 µL culture + 900 µL LB.
5. Measure OD600 using the spectrophotometer.
6. Recorded OD600 (1:10 dilution): 0.3
7. Calculate estimated undiluted OD600:
8. OD600(original) = 0.3 × 10 = 3.0

**Preparation of Large Culture**

1. Add 50 mL of sterile LB broth to the autoclaved 500 mL flask.
2. Inoculate with 1 mL of overnight culture.
3. Incubate at 37°C with shaking.

**Measuring OD600**

1. Obtain a clean cuvette.
2. Add 900 µL LB broth → place in spectrophotometer → align arrow on cuvette with arrow on machine → press left button to zero (0.000).
3. Discard LB.
4. Add 900 µL of culture (or appropriate dilution) to the cuvette.
5. Insert in same orientation and press right button to record OD600.
6. Record OD600 at regular time intervals.

## References

[1] “Addgene: Protocol - How to Design Primers.” Accessed: Oct. 06, 2025. [Online]. Available: <https://www.addgene.org/protocols/primer-design/>

[2] B. Maier, “Competence and Transformation in *B. subtilis*,” *Curr. Issues Mol. Biol.*, vol. 37, pp. 57–76, 2020, doi: 10.21775/cimb.037.057.

[3] M. Jakobs and F. Meinhardt, “What renders *Bacilli* genetically competent? A gaze beyond the model organism,” *Appl. Microbiol. Biotechnol.*, vol. 99, no. 4, pp. 1557–1570, Feb. 2015, doi: 10.1007/s00253-014-6316-0.

[4] L. Vojcic, D. Despotovic, R. Martinez, K.-H. Maurer, and U. Schwaneberg, “An efficient transformation method for *B. subtilis* DB104,” *Appl. Microbiol. Biotechnol.*, vol. 94, no. 2, pp. 487–493, Apr. 2012, doi: 10.1007/s00253-012-3987-2.

[5] J. Caro-Astorga et al., “SubtiToolKit: a bioengineering kit for *B. subtilis* and Gram-positive bacteria,” *Trends Biotechnol.*, vol. 0, no. 0, Mar. 2025, doi: 10.1016/j.tibtech.2025.02.004.

[6] K. B. Falkenberg, C. H. Rollan, M. Rennig, A. B. Bertelsen, and M. Norholm, “Transformation of heterolous DNA in *B. subtilis*,” Oct. 2020, Accessed: Sep. 21, 2025. [Online]. Available: <https://www.protocols.io/view/transformation-of-heterolous-dna-in-bacillus-subti-bdmti46n>

[7] M. S. Payne and E. N. Jackson, “Use of alkaline phosphatase fusions to study protein secretion in *B. subtilis*,” *J. Bacteriol.*, vol. 173, no. 7, pp. 2278–2282, Apr. 1991, doi: 10.1128/jb.173.7.2278-2282.1991.

[8] G. Wang et al., “A new potential secretion pathway for recombinant proteins in *B. subtilis*,” *Microb. Cell Factories*, vol. 14, no. 1, p. 179, Nov. 2015, doi: 10.1186/s12934-015-0374-6.

{% endblock %}

{% extends "layout.html" %}
{% from "components/hero-macro.html" import custom\_hero %}
{% from "components/image-card-macro.html" import image\_card %}
{% block title %}Results{% endblock %}
{% block sidebar %}

![Close sidebar](data:image/svg+xml... "Close side bar")

## Table of Contents

* [Introduction](#introduction)
* [AMP Selection](#AMP-selection)
* [Phylogenetic Analysis](#Phyto-DB)
* [Basic Parts](#basic-parts)
* [PhoA Secretion Assay](#phoA-assay)
* [Dual Culture Benchmark](#benchmark-growth)
* [Competition Assay](#constructs-competition)
* [System Versatility](#sumo)
* [Conclusion](#conclusion)
* [References](#references)

{% endblock %}
{% block page\_content %}
{% set results\_cards = [
{
'image': 'https://static.igem.wiki/teams/5691/results/plasmid.webp',
'title': 'Successful Engineering',
'text': 'Successfully engineered *Bacillus subtilis* with plasmids allowing produce and secrete antimicrobial peptides.'
},
{
'image': 'https://static.igem.wiki/teams/5691/results/benchmark.webp',
'title': 'Dual-culture assay optimization',
'text': 'We described and optimized a dual-culture plate assay to evaluate the antimicrobial activity of engineered *B. subtilis* against *Phytophthora* species.'
},
{
'image': 'https://static.igem.wiki/teams/5691/results/cecropin.webp',
'title': 'In-silico AMP selection',
'text': 'We screened public databases for AMPs against *Phytophthora* species.'
},
{
'image': 'https://static.igem.wiki/teams/5691/results/figure-4-real.webp',
'title': 'Secretion reporter assay optimization',
'text': 'We described an assay to evaluate the secretion efficiency in *B. subtilis*, using PhoA as a reporter protein.'
}
] %}
{{ custom\_hero(
subtitle="Project / Results",
title="Results",
intro="Comprehensive results from our PhytoBlock biocontrol system development, demonstrating successful engineering of *B. subtilis* and validation of our antimicrobial agent *Phytophthora* species.",
cards=results\_cards
) }}

## Introduction

The PhytoBlock project aimed to develop a sustainable biocontrol solution against *Phytophthora* species that cause devastating black pod disease in cocoa plants. Through systematic engineering and testing, we have achieved significant milestones in creating a functional microbial biocontrol agent based on *Bacillus subtilis*.
Our goals is to develop a sustainable biocontrol solution against *Phytophthora* species that cause the devastating black pod disease in cocoa plants. Through multiple cycles of engineering and testing, we have achieved significant milestones in creating a microbial biocontrol agent based on *Bacillus subtilis*.

Our results demonstrate successful implementation of our dual-input biosensing system, effective production and secretion of antimicrobial peptides, and validation of their activity against target pathogens. This section presents comprehensive findings from our laboratory investigations, including molecular characterization, functional validation, and preliminary efficacy studies.

## Finding suitable AMPs to inhibit *Phytophthora* growth

Upon reviewing the antimicrobial peptides (AMP) database, as described in the Model page, we chose the following AMPs as leads:

* Two vicilin-like peptides from the macadamia tree (*Macadamia integrifolia*) (MiAMP2-2 and MiAMP2c-2). These AMPs have been reported to suppress hyphal growth in oomycetes like *P. cryptogea* [1]. Therefore, these two candidates were selected for wet lab validation.
* Two European spindle (*Euonymus europaeus*) proteins. Ee-CBP is found on leaves, while a closely related isoform is produced in the bark. These lectin-like peptides have been shown to bind cell wall carbohydrates in *P. cryptogea*, thus disrupting growth of the organism. Their activity has been reported independently for different isolates, providing support that extends beyond a single strain and justifying inclusion for in vitro testing [2][3].
* Two short antifungal peptides (Antifungal peptide 1 and Antifungal peptide 2) originating from the rubber tree (*Eucommia ulmoides*), with documented effects on *P. infestans*. These peptides are small, cationic and have traits associated with membrane permeabilization and rapid killing of pathogens. They have confirmed activity against *Phytophthora* [4], and since *P. infestans* and *P. palmivora* share similar membrane lipid compositions and cell wall polysaccharides, these AMPs have been shortlisted for further testing.
* Lucimycin (LSer-AFP), derived from the green bottle fly (*Lucilia sericata*), complements our predominantly plant-based list with a defense molecule derived from an invertebrate. Although it also exhibits some antibacterial activity, its antifungal potency and resistance to proteolytic degradation are particularly relevant, with reported activity against *Phytophthora parasitica* [5]. Its mode of action likely involves membrane or cell-envelope disruption, which supports our rationale for expecting activity against *Phytophthora palmivora*.
* EP-20, derived from *Xenorhabdus budapestensis*, was included because antimicrobial peptides from entomopathogenic bacteria often display broad antifungal activity. EP-20 has been reported to inhibit *Phytophthora capsici* [6]. Its short length, cationic charge, and proven efficacy against oomycetes make it a suitable candidate for expression and screening.
* Cecropin from the tsetse fly (*Glossina morsitans*). Although cecropins are best known for their antibacterial activity, this peptide has also been shown to inhibit *Phytophthora infestans* [7].

Taken together, the nine selected AMPs represent a consistent trend in our database search: each peptide is documented in public repositories with primary literature support, demonstrates at least one reported activity against a *Phytophthora* species, and possesses a plausible mechanism of action that could extend to *Phytophthora palmivora*. Preference was given to AMPs with either inhibitory effects against *Phytophthora*, mechanisms involving interaction with cell wall components or membrane disruption.

In the future, we aim to gain a deeper understanding of the physical properties of these peptides, enhance efficacy and improve stability through protein engineering.

## Phylogenetic analyses of the *Phytophthora* genus allow us to refine predictions of AMP activity

Our *Phytophthora* genome database covers 174 different species. Most of them have a hemi-biotrophic lifestyle and they are found all across the world. Common hosts are cocoa trees, tomato plants, soybean, and citrus. The combination of conserved lifestyle and biology makes envelope-targeting AMPs especially attractive. As a result, mechanisms that bind β-glucans or destabilize membranes are likely to maintain efficacy across the genus.

We have taken particular interest in three *Phytophthora* species: *P. infestans*, *P. cryptogea*, and *P. palmivora*. While our main focus is on *P. palmivora* affecting cocoa trees, *P. infestans* and *P. cryptogea* are naturally present in Belgium. Consequently, these local species can serve as suitable models for evaluating the effectiveness of our antimicrobial peptides (AMPs) in case it is impossible to obtain *Phytophthora palmivora*.

The *P. infestans* T30-4 genome (228.5 Mb, 51% GC) is large and repeat-rich, making it particularly susceptible to AMPs that target conserved membrane and cell wall components [8]. This supports our selection of cationic, amphipathic, and lectin-like peptides effective against *P. infestans*, *P. cryptogea*, and related species.

*P. cryptogea* CBS 418.71 (63.8 Mb, 52.5% GC) is a hemibiotrophic species with a broad host range **[9]**. Several of our candidate AMPs, including vicilin-derived peptides such as MiAMP2 and carbohydrate-binding proteins like Ee-CBP, have demonstrated inhibitory effects on this species, supporting its suitability as a potential model for future testing of our system.

*P. palmivora* sbr112.9 (107.8 Mb, 49% GC) is a hemibiotrophic species that primarily infects *Theobroma cacao* and is distributed across major cocoa-growing regions [10]. Its β-glucan– and cellulose-rich cell wall aligns well with the mechanisms of our selected AMPs, including β-glucan–binding lectin-like peptides and short, cationic amphipathic helices. Together with *P. infestans* and *P. cryptogea*, these reference genomes represent key traits relevant to our research, increasing the likelihood that our chosen AMPs will demonstrate effective inhibition against phylogenetically or functionally similar strains.

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-1.webp",
image\_alt="Phytophthora genomes results",
footer\_text="Figure 1: Pairwise whole-genome synteny between *Phytophthora infestans*, *P. cryptogea*, and *P. palmivora*"
) }}
Using MUMmer4 **[11]** genome alignment system, we conducted a comparative homology study with the previously listed reference genomes. The whole-genome alignment results indicated that *P. infestans*, *P. cryptogea* and *P. palmivora* are closely related at the genomic level.

The MUMmerplot dotplot illustrates the genomic alignment between two species, highlighting regions of sequence similarity and structural variation. The prominent blue diagonal line from the bottom left to the top right indicates a strong level of synteny, suggesting that large portions of the two genomes are conserved in both sequence and gene order. This reflects a close evolutionary relationship or limited genomic rearrangement between the species.

The presence of scattered purple and blue dots off the main diagonal signifies local sequence similarities that are either rearranged, inverted or fragmented due to assembly differences. Therefore, the plots support a high degree of homology between the species, with some evidence of genomic inversions and structural variations.

The conserved synteny and the overlap in predicted gene content all point to these three species sitting near one another within *Phytophthora*, differing primarily at the margins of their genomes rather than in their backbone.

## Nine sequence-optimized basic parts expand the SubtiToolKit for production of AMPs in *B. subtilis*

Thanks to the kind support of Prof. Joaquin Caro-Astorga, we obtained access to the SubtiToolKit (STK). The use of STK enables efficient assembly of the desired constructs by using Golden Gate Assembly. This technique utilizes specific Type IIS enzymes to cut and assemble in a modular way. Acquired STK strains contain plasmids encoding various promoters, RBSs, reporter proteins, secretion tags and terminator sequences optimized for use in *B. subtilis*, which allowed us to streamline our cloning approach. Each STK part is equipped with specific adapters, whose sequence depends on the type of the part (Figure 2). Variation is possible for the CDS part, which can be split into Cx, Cy and Cz regions, allowing assembly of proteins with different N- and C-terminal tags.

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-2.webp",
image\_alt="STK",
footer\_text="Figure 2: SubtiToolKit syntax allows for hierarchical assembly of parts and construct for *B.subtilis*. Copied from [20]"
) }}

We aimed to expand the STK by incorporating novel parts – encoding selected AMPs, a reporter PhoA protein and two fusion tags, aimed to facilitate protein purification – into STK syntax and testing their performance in B. subtilis. All basic parts were codon-optimized for efficient expression in B. subtilis using GenSmart™ Codon Optimization tool. To ensure compatibility with STK and compliance with iGEM registry guidelines, we avoided BsaI, SapI, BbsI and BsmBI restriction sites in the design.

The parts were synthesized as gBlocks by Twist or IDT. To ensure efficient Golden Gate Assembly (GGA) of the final constructs, the parts were pre-cloned into level 0 plasmids (comparable to basic parts) and stored in *E. coli* DH10B. Reaction conditions were optimized (see the [Engineering Cycle 1](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D#engineering-cycle-1) for more information). The final GGA cloning protocol is described on [the Experiments page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D#golden-gate-assembly). After transformation, non-fluorescent colonies were verified by colony PCR, and plasmids from positive clones were isolated and sent for sequencing.

By the end of the project, we successfully cloned the following basic parts into level 0 plasmids:

Cy:

* MiAMP2-2 – [BBa\_25DO0ICK](https://registry.igem.org/parts/bba-25do0ick)
* MiAMP2c-2 – [BBa\_258YJPQ3](https://registry.igem.org/parts/bba-258yjpq3)
* Ee-CBP-Leaves –  [BBa\_25OQ2MUK](https://registry.igem.org/parts/bba-25oq2muk)
* Antifungal peptide 1 - [BBa\_25LAH38O](https://registry.igem.org/parts/bba-25lah38o)
* Antifungal peptide 2 - [BBa\_25CSAZHD](https://registry.igem.org/parts/bba-25csazhd)
* Lucimycin – [BBa\_25N58AWT](https://registry.igem.org/parts/bba-25n58awt)
* EP-20 – [BBa\_25DO0ICK](https://registry.igem.org/parts/bba-25do0ick)
* Cecropin – [BBa\_25CH9T79](https://registry.igem.org/parts/bba-25ch9t79)

Cx:

* HIS-Thioredoxin-TEV – [BBa\_25FN8UNC](https://registry.igem.org/parts/BBa_25FN8UNC)
* HIS-SUMO – [BBa\_25FN8UNC](https://registry.igem.org/parts/BBa_25FN8UNC)

Cx/Cz:

* PhoA from *E. coli* – reporter protein

Thus, we expanded the STK with parts codon-optimized for production in *B. subtilis*, enabling production of seven different AMPs, SUMO and thioredoxin fusion tags, and a reporter PhoA gene.

## Codon-optimized alkaline phosphatase (PhoA) secretion reporter in *B. subtilis*

To visualize the effects of xylose induction and provide a platform for testing different secretion tags, we designed a reporter system employing alkaline phosphatase (PhoA) from *E. coli* as a reporter gene. The amino acid sequence was downloaded from the GenPept database (accession number NP\_414917.2) and optimized as described above. To lower the complexity of the GGA reaction, the designed overhangs allowed assembly with the secretion tag and terminator parts, omitting the Cz linker part. We assembled and tested six constructs differing in the type of Cx tag and RBS. All designs included the xylose promoter part (STK161\_pSTK-0A-XylR-Pxyl) and a strong terminator (STK080\_pSTK-0D-bkdB Cold double terminator). The constructs contain either Apr or SacB secretion tag or a Cx linker. Additionally, we tested two RBS sequences.

PhoA has been used previously as a reporter of secretion in *B. subtilis* [12], but, to our knowledge, a detailed secretion assay protocol for this chassis has not been described by any previous iGEM team. Our goal was to design a [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) that can be easily reproduced and adapted by future teams.

In our pilot assay, we employed the following controls:

* Purified bovine alkaline phosphatase from Promega – positive control
* Pellet and supernatant samples from *B. subtilis* 168 WT – negative control

Moreover, we baseline-corrected our measurements with the growth media (LB).

Out of all engineered strains, only *B. subtilis* transformed with pPB026 showed clear accumulation of product in the supernatant sample. In that construct, PhoA was fused with the Apr tag. This secretion tag is documented to permit secretion of PhoA in *B. subtilis* [11]. The efficiency of secretion is notably high, with the signal from the supernatant over 10-fold higher than that of the cell lysate sample (**Figure 3**).

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-3.webp",
image\_alt="phoA assay results",
footer\_text="Figure 3: One of the engineered *B. subtilis* strains efficiently secretes active alkaline phosphatase. OD410 values were measured in 3 replicates in 3 individual assay runs each. Statistics performed by a two-way ANOVA with Sidak multiple comparisons test. \*\*\*\* p lower than 0.0001."
) }}

To assess the reproducibility of the assay, we plotted the increase of OD410 over time for the strain transformed with pPB026, measured in three assay runs (**Figure 4**). The results were highly similar across the first two repeats. We assume that slight modifications to the protocol in the third repeat (described in the [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)) caused the higher variability observed.

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-4-real.webp",
image\_alt="PhoA assay reproducibility results",
footer\_text="Figure 4: The activity of Apr-PhoA in the supernatant is over 10-fold higher than in the corresponding amount of the cellular lysate across assay runs. Each point represents the mean measurement of the three replicates measured at the particular time in each assay run. Dotted lines are a representation of the error bars (SD)."
) }}

Future prospects:

* Further optimization of the assay – the goal would be to observe the whole saturation curve, allowing to incorporate specific models and measurements of the kinetic properties of secretion tag-PhoA fusions. Moreover, we need a way to relate the enzymatic activity to the absolute amount of the enzyme in the medium.
* The current protocol involves long incubation times. Perhaps the modification of the protocol (induction with higher amount of xylose, addition of higher concentration of the substrate) would increase the sensitivity of the assay, allowing to shorten the incubation time post-induction.
* The first paper describing the use of PhoA as a secretion reporter in *B. subtilis* mentions the prospect of creating tri-partite fusions to analyze the secretion tag preference of the protein of choice by measuring the activity of PhoA fused to that protein [11]. The main end goal of this assay is to evaluate this approach to measure the secretion of AMPs. First, we need to adapt a protocol allowing to confirm the presence of the AMP in the supernatant sample upon secretion via a particular secretion tag (SDS-PAGE electrophoresis, HPLC), then compare the results of such assay to the phosphatase activity of the secretion of the fusion of such AMP with the same secretion tag and PhoA.

## Our plate-based dual culture assay setup allows standardization of microbial growth measurements

We adapted and described one of the classic dual-culture plate assay formats, wherein the mycelium plug with *Phytophthora* is placed in the centre of the plate, surrounded by three bacterial colonies. Secretion of anti-oomycete compounds by tested bacteria inhibits the growth of *Phytophthora* mycelia near the bacteria, leading to the formation of “halos”.

To standardize the conditions of the assay, we measured the growth of both tested *Phytophthora* strains and *B. subtilis* 168 on five different solid media: carrot agar (CA), pea sucrose agar (PSA), Lysogeny broth agar (LB), V8-juice agar (V8) and potato dextrose agar (PDA). This was tested at two different temperatures, 27°C and 30°C. In each test run, we included 4 replicates of each microorganism growing on a particular type of media.
Cost analysis suggests that our biocontrol agent could be economically viable for smallholder farmers, particularly when considering reduced environmental impact and potential for resistance management compared to chemical alternatives.

In general, we observed better growth of *P. capsici* than *P. palmivora* on all tested media except PSA (Figure 5). The overall effect of temperature on incubation was not significant (p = 0.2102 in 3-way ANOVA), although it appeared to impact *P. palmivora* growth on PSA and PDA (Figure 5).

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-5.webp",
image\_alt="Benchmark growth results",
footer\_text="Figure 5: Tested strains of *P. palmivora*  and *P. capsici* exhibit different media preferences. The end-point total area of each *Phytophthora* colony grown after 4 days was measured by the ReShape Botech Imaging Device in two temperature variants. In each assay run, 4 replicates were measured. Statistics performed by 2-way ANOVA with Sidak multiple comparisons test. Significance illustrated according to GP p-value style (\*\* p=0.001, \*\*\* p=0.0002, \*\*\*\*p below 0.0001)"
) }}

Analysis of the growth data indicated that pea sucrose agar (PSA) at 27 °C supported the most consistent and robust growth for both organisms (Figure 6).

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-6.webp",
image\_alt="Benchmark growth curves",
footer\_text="Figure 6: Growth curves of *Phytophthora* species on pea sucrose agar (PSA) at 27°C."
) }}

Future prospects:

* Gathering of the measurements of the *B. subtilis* media preference needs optimization (Fig. 7). Despite plating the same amount of bacterial cultures, diluted to a particular optical density, plate-to-plate variation between the technical replicates in the same assay is high. Perhaps the assessment of the growth curves of bacteria growing in the liquid media would be a useful indicator of the media preference.
* Ideally, we would repeat the growth assay at least twice per temperature variant to increase our confidence in the collected results.

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-7.webp",
image\_alt="Figure 7 bacillus",
footer\_text="Growth media and temperature affect the morphology and the growth properties of *B. subtilis*. The upper panel represents the number of colonies formed on their respective media, throughout the incubation period during incubation at 27- and 30 degrees. The error bars represent SD."
) }}

After establishing the initial conditions of the assay, we conducted a test dual culture experiment, where  *Phytophthora* strains were cultured with  *B. subtilis* strains expressing GFP upon induction with xylose. This served as a validation step for the final assay.

During this stage, we tested two parameters:

1. Whether different xylose concentration improves protein expression without negatively impacting *Phytophthora* growth.
2. The optimal volume of bacterial suspension required per assay plate.

We observed that GFP fluorescence was much stronger at 2% xylose compared to 1% xylose, confirming that induction efficiency improved, but at the cost of negatively impacting the growth of *P. palmivora* (Figure 8). Due to this effect we decided to use 1% xylose in our final competition assay experiment.

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-8.webp",
image\_alt="Benchmark growth with xylose results",
footer\_text="Figure 8: Xylose concentration impacts the induction of the heterologous expression of GFP in the engineered *B. subtilis*strain and the growth of *P. palmivora* strains. The images were collected at the end of the incubation period (27°C 4 days), GFP filter of the machine was set to strength 5.0. (A) B. subtilis GFP production at 1% xylose (B) *B. subtilis* GFP production at 2% xylose (C) *P. palmivora* growth in 1% xylose (D) *P.palmivora* growth in 2% xylose."
) }}

Future prospects:

* Further evaluation of the xylose content in the assay media would allow us to pinpoint the highest concentration of xylose that has an effect on the induction of the Pxyl promoter without inhibiting *P. palmivora* growth.

## Constructs allowing for the secretion of two AMPs can be easily transformed to *B. subtilis*

The main objective of the project was engineering and evaluation of the activity of AMP-secreting *B. subtilis* strains. In our assemblies, we used parts from the SubtiToolKit alongside the basic parts we incorporated into the STK syntax. We followed the experimental procedures described in the STK paper with slight modifications (see Experiments page for detailed protocols).

The initial design was straightforward: expression of the AMP–secretion tag fusion was regulated by the xylose promoter. To mitigate potential toxicity effects in *E. coli*, we included the “E. coli Expression Blocking Device” RBS part from STK in a variant of the assembly. Additionally, we tested two secretion tags (Apr and SacB) and a Cx linker part (non-secreting) to account for different secretion-tag preferences of the tested AMPs.

Assembly of the final constructs allowing for the production and secretion of AMPs proved to be more difficult than anticipated. The troubleshooting process is described extensively in the [Engineering Cycle 4](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D#engineering-cycle-4)
. In the end, we successfully assembled plasmids allowing for the xylose-dependent production of the Vicilin-like antimicrobial peptide 2-2 and cecropin fused to the SacB secretion tag or Cx linker, as confirmed by sequencing data. Those constructs were then transformed to B. subtilis 168 strain. Engineered bacteria were then evaluated for their ability to inhibit the growth of *Phytophthora*.

We did not see formation of mycelia-devoid halos around the *B. subtilis* colonies, which would signify inhibitory activity of the engineered strains. Interestingly, we observed two different mycelial growth behaviors depending on incubation with specific *B. subtilis* strains. *P. palmivora* appeared to exhibit faster growth (compared to the culture cultivated alone) when cultured with *B. subtilis* strains expressing tested AMPs with a SacB linker. In contrast, *P. capsici* seemed to grow equally fast when incubated with strains carrying SacB–AMP fusions or grown alone, but its growth when cultured with *B. subtilis* expressing GFP was slower.

The time constraints did not allow us to repeat the experiment. We believe that the observed results might be related to a different amount of plated mycelia, which could allow for faster *Phytophthora* growth independent from the effect of the treatment.

We observed that the morphology of *B. subtilis* colonies differed from plate to plate (Figure 9). Strains transformed with a secretion tag and MiAMP2-2 (Figure 9C) or cecropin (Figure 9D) appeared “granular,” as if bacterial growth was affected, perhaps by the produced AMPs. This would signify a potential toxic effect of the selected peptides on engineered bacterial cultures and the need to screen for new AMPs or explore fusion tags protecting the production host.

{{ image\_card(
image\_src="https://static.igem.wiki/teams/5691/results/figure-9.webp",
image\_alt="B. subtilis morphology results",
footer\_text="Figure 9: *B. subtilis* impact on growth of (A) *P. palmivora* and (B) *P.capsici* throughout the competition assay, images were analyzed manually by using Fiji (ImageJ). Lower panels (C,D) Colony morphology of *B. subtilis* 168 expressing MiAMP 2-2 and Cecropin."
) }}
If the production of the AMPs affects the growth of *B. subtilis*, we hypothesize that perhaps the observed effect on the growth speed of *Phytophthora* may be due to the release of other compounds by *B. subtilis* under stress to the medium.

Future prospects:

* To confirm whether the observed differences are biologically significant, we aim to repeat the assay and report better on the amount of mycelia used in the assay.
* Moreover, we need to optimize the method of data collection. ReShape Biotech Imaging Device cannot properly track the growth of *Phytophthora* over time in presence of three colonies belonging to different species. We measured the changes in the area by manually analyzing the pictures of the plates with Fiji (ImageJ) software.
* We hypothesize that the observed effect – if true – is not directly related to the ability of tested AMPs to affect the growth of Phytophthora. We want to transform our constructs to another *B. subtilis* strain lacking proteases/genome minimized to ensure the stability of the AMPs. Additional protocols involving visualization of AMPs on SDS-Page gels or HPLC, without relying on the functional assays to confirm the peptide production, are also in plans.
* We aimed to keep our cloning strategy as versatile as possible – thus, the assembly method enables the substitution of the secretion tag with a SUMO fusion tag. This will allow us to purify the peptides and measure their effect on the *Phytophthora* strain directly to assess their specificity.

## Versatility of our system: from biocontrol strain engineering to peptide purification

Anticipating problems with assembly of constructs containing AMP–secretion tag fusions, we included 6xHis-SUMO and 6xHis-thioredoxin Cx parts in our basic parts repertoire. This allowed us to substitute the secretion tag for a fusion tag in the GGA reaction when needed.

Engineering bacteria to produce peptide–SUMO fusions has proven a successful strategy to obtain significant yields of numerous AMPs [13–17]. This approach is usually used to purify AMPs from cell lysates or culture media (as opposed to using the engineered strain directly as a biocontrol agent), as attachment of SUMO may interfere with inhibitory effects. Conversely, fusion tags can lower peptide toxicity to the production host. As we struggled to engineer *B. subtilis* strains secreting AMPs, assemblies enabling purification of the peptides would allow us to assess their activity on the tested *Phytophthora* spp.

We assembled the construct bearing the sequence of 6xHis-SUMO–cecropin and transformed it to *B. subtilis* 168. Interestingly, we did not observe growth of transformants in LB liquid medium. Production of SUMO fused with a cecropin AD variant in *B. subtilis* has been reported in the literature [17, 18]. Thus, we believe the lack of growth is most likely the result of technical issues or contamination of the batch of competent *B. subtilis* cells.

Future prospects:

* Persisting issues regarding cloning and evaluation strategies reinforces the need to quality-check all used sources (chemocompetent *E. coli* and *B. subtilis* cells, reagents, STK strains, media) to exclude contaminations
* We would like to test the effect of the growth conditions (rich/minimal medium, growth temperature, shaking conditions) on the performance of the strain
* Lastly, we could test other fusion tags like 6xHis-thioredoxin-TEV fusion for their ability to increase the yield of the produced AMP. Several carrier proteins have been used to increase the yield of producing peptides in bacteria, each characterized by its own advantages and disadvantages [19].

## Conclusion

We successfully cloned and assembled multiple AMP constructs in *Bacillus subtilis* and developed benchmark assays to identify optimal dual-culture assay conditions. Although functional assays revealed noticeable morphological changes in *B. subtilis* expressing AMPs, no inhibition zones or significant effects on *Phytophthora* growth were observed. These findings suggest that our AMP library may need to be expanded and refined to include more effective candidates. Overall, the results provide a solid foundation for future work, emphasizing the need for further optimization of construct expression and secretion to achieve consistent antifungal activity.

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