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Lab / Measurement
Measurement
Accurate measurement is a cornerstone of scientific inquiry, which is why we strived for the highest possible precision in all our work.
Summary
We made a robust measurement framework to ensure accuracy, reproducibility, and comparability of our experimental results.
Plate-based dual-culture assay
The plate-based dual culture assay was used to evaluate the inhibitory effect of our engineered B.subtilis strains against Phytophthora spp
Secretion assay setup for our engineered bacteria
The secretion assay using the PhoA was designed to confirm the secretion of our AMPs by our engineered B.subtilis strains.
Introduction
The goal of the wet lab component of the project was to engineer
B. subtilis strains to produce and secrete AMPs that inhibit the
growth of Phytophthora that is responsible for causing black pod
disease. Hence, we obtained two species known to infect cocoa plants: P.
palmivora and P. capsici. We established and described an experimental
pipeline to verify that all Phytophthora and bacterial strains are able
to grow on the plates, that secretion was functional and optimized, and
to test the efficiency of the chosen AMPs plate-based format. This
involves: Standardization of the amount of plated
B. subtilis allowing consistency between each assay Optimization
of the culture conditions of the tested strains Execution of the
experimental setup in a competition assay Additionally, we described an
assay allowing to study the secretion in B. subtilis using PhoA
as a reporter protein. As this pipeline is well-described and
reproducible, we offer a guideline for future iGEM teams intending to
work with B. subtilis or other potential biocontrol strains
Plate based Dual Culture Assay
Background Information
Plate-based dual culture assays are a common technique used to measure the antagonistic effect of various microbial strains on the growth of Phytophthora. Their main advantages are: No need for specialized infrastructure (only requires an incubator) Straight forward data analysis. The general idea behind the assay is simple. Microorganisms are cultured in a chosen temperature for a specific period. Then, the inhibitory effect of the antagonistic strain is measured and reported. There is some diversity in the plate layouts used by different groups (Fig. 1). They differ in the placement of the mycelial plug (either in the center of the plate or at the side), and the amount or the shape of the plated antagonist (either a droplet or a line around or at the side of the mycelial plug). The layout of the plate influences the type of measurements used to measure inhibition. If the mycelial growth is even in all dimensions, reporting on the differences in the radius of the treated and untreated Phytophthora provides enough information to report an effect [1]. Similarly, using software like ImageJ allows to measure the area of the Phytophthora colony rather than its radius after incubation. This approach was used to quantify the growth of Phytophthora. Alternatively, the bacterial sample can be placed next to the mycelial plug, here the radial growth is measured and the difference in the radius of Phytophthora growing towards the antagonist and towards the control sample on the same plate can be used to report inhibition [2]. Another popular approach is placing the mycelial plug at the edge of the plate and plating the bacteria in a line at a certain distance [3]. We chose to use our method, which is also widely adopted, because our secondary PI, Barbara, has extensive experience conducting competition assays with Phytophthora species and could provide guidance if any issues arose. The growth medium, temperature and the incubation time must be optimized for each assay to account for different growth preferences of the tested strains of Phytophthora. While some papers evaluate the performance of Phytophthora strains on different culture media, we noticed that it is not a common practice to document the argumentation for the choice of a particular media in articles describing dual culture assays with Phytophthora
Measurement of B. subtilis Growth
The goal of our initial experiment was to relate OD600 measurements to CFU number obtained from the B. subtilis grown in LB medium. Our goal was to establish at which value of OD600 we obtain countable number of B. subtilis colonies, allowing for reliable comparison across the different media tested.
Procedure
A standard protocol, described in detail on the Experiments page.
Comparing the growth properties of the tested strains in different conditions
Procedure
We began our optimization efforts by measuring mycelial and bacterial growth on different media, across two temperatures: 27 °C and 30 °C. We tested the following media that are frequently used to culture Phytophthora species: V8-agar, potato dextrose agar (PDA), carrot agar (CA), pea sucrose agar (PSA). Additionally, we tested the growth of the strains on LB agar as a control for B. subtilis. We added the media to 6-well plates to increase the throughput of the assay. In each assay run, we included 2 plates with the specific medium type, each containing 2 wells with a given microorganism. Therefore, each assay contained 4 replicates of the particular growth condition for each strain. To measure Phytophthora growth, we placed a mycelium plug (collected using a borer from the edges of the growing stock) in the center of the plate. To assess the media preference of B. subtilis, we spread 50 µL of the 10-5 dilution evenly on a plate to obtain around 50 colonies/plate. This was based on optical density, measured in an assay, and was supported by literature. We collected the data until the mycelia covered the whole plate for one of the tested conditions (4 days).
Data Analysis
For Phytophthora samples, we analyzed 2 types of data Area [mm2] of the plate covered by mycelia Increase of the area [mm2] covered by mycelia over time, fitted into Gompertz equation For B. subtilis samples, we measured the number of colonies formed on each plate. We used the built-in analysis software of the ReShape Biotech Imaging Device to obtain the area measurements. For those that do not have the imaging device, this growth can be tracked by periodically taking pictures and analyzing them with ImageJ [4].
Dual-culture assay assessing the antagonistic effect of our engineered strain
Procedure
We plated the mycelial plug in the center of the plate. Then, we positioned three droplets of B. subtilis culture of an OD600 = 0.4, 1.5 cm from the center of the plate (Fig.2). We included the following controls: Phytophthora growth control: only Phytophthora plated in the middle of the plate Baseline growth: with B. subtilis and the Phytophthora species. Phytophthora was plated in the middle of the plate, surrounded by 3 droplets of B. subtilis 68 that produced a protein (GFP) with no activity against Phytophthora. These plates were also incubated for 4 days.
Data Analysis
We collected the pictures of the plates every 30min throughout the duration of the assay, using the ReShape Biotech Imaging Device. We collected Phytophthora growth area [mm2] over time: we measured the area using Fiji (ImageJ) software from pictures in 2-hour intervals.
Results
Our preliminary data shows some differences in the growth curves of the assayed strains. More repeats of the assay are needed to increase the strength of our conclusions. Refer to the results page for preliminary hypotheses.
PhoA Secretion Assay
To the best of our knowledge, no previous iGEM team has described a detailed protocol or built a dedicated PhoA-based secretion assay for B. subtilis. By implementing this approach, we aim to establish a reproducible and quantitative framework for characterizing secretion tags in B. subtilis, leveraging the well-documented stability, specificity, and assay simplicity of the PhoA reporter system.
Background information
We designed this preliminary assay to evaluate secretion tag efficiency by substituting our AMP sequence with alkaline phosphatase (PhoA), whose activity can be quantified by measuring the conversion of the substrate pNPP (para-nitrophenylphosphate) into the yellow product pNP (para-nitrophenol). Previously, PhoA has been widely used as a reporter of secretion in B. subtilis, a lab demonstrated that the system showed alkaline phosphatase activity which strictly depends on secretion, since intracellular PhoA remains inactive in the reducing environment of the cytoplasm [5]. This property makes PhoA a robust, secretion-dependent reporter for B. subtilis, enabling straightforward quantification of export efficiency through simple colorimetric or spectrophotometric assays. In addition to bacterial systems, secreted alkaline phosphatase (SEAP) has also been developed and successfully applied as a reporter protein in mammalian and eukaryotic models, including CHO-K1 cells, where it allows noninvasive monitoring of gene expression through detection of enzyme activity in the culture medium [6]. SEAP was less efficient than M-Luciferase in eukaryotic cell lines but could still prove useful in niche experimental requirements.
Preparation of Cell Lysate and Culture Supernatant Samples
Procedure
Overnight cultures of the transformed B. subtilis strains were first prepared. Each culture had a total volume of 5 mL, and three biological replicates are prepared for each construct to exclude possible biological differences between samples. The diluted cultures were incubated at 37 °C, under shaking condition until they reached an OD₆₀₀ ≈ 1, after which 1% xylose was added, to induce expression of phoA. Cultures were further incubated for 3 hours to allow sufficient induction. Following centrifugation, the supernatant was collected for analysis, and the cell pellets were resuspended in 5 mL lysis solution (0.01% SDS, 1 mg/mL lysozyme). Lysates were centrifuged at 5000 G for 15 minutes at room temperature, and the resulting supernatant was collected as the cell lysate fraction.
Preparation of the Reaction Mixture and Measurement of PhoA signal
Procedure
For each reaction, 100 μL of substrate buffer was added to a well of a 96-well microplate, followed by 100 μL of the bacterial sample (either supernatant or lysate). The enzymatic reaction was monitored at 410 nm (OD₄₁₀) using a plate reader, with measurements taken every 30 seconds, for 30 minutes at room temperature, with shaking. Purified bovine alkaline phosphatase (Promega) was used as a positive control, while pellet and supernatant samples from B. subtilis 168 WT served as negative controls. All measurements were baseline-corrected using the LB growth medium.
Data Analysis
Data was analyzed by plotting OD₄₁₀ values over time to monitor the accumulation of the colored product (p-NP), which reflects alkaline phosphatase activity. End-point OD₄₁₀ values could also be used to compare the enzymatic activity between the culture supernatant and the cell lysate samples, providing an estimate of secreted versus intracellular PhoA activity.
Results & Discussion
The secretion efficiency was remarkably high, as indicated by a more than tenfold increase in the signal detected in the supernatant compared to the cell lysate sample (Fig. 4).
iGEM Best Measurement Criteria
For your convenience, we summarize how PhytoBlock addresses all iGEM Best Measurement Criteria:
Our measurement approach is fully reproducible by other teams. The plate-based dual culture assay was performed using the ReShape Biotech imaging device, but the same measurements can be replicated manually. Any future team can follow our detailed protocol, take images at the same intervals, and analyze them using accessible software such as ImageJ. Likewise, the B. subtilis secretion assay is described step-by-step, allowing other teams to repeat it independently with reliable results.
We have thoroughly documented all experimental procedures. The full protocols for both the secretion assay and the plate-based dual culture assay are clearly presented in the Experiments tab. Our underlying scientific reasoning, design choices, and optimization steps are further detailed in the Notebook and Engineering tabs. This ensures transparency and reproducibility for future users.
Our measurement framework is designed to be broadly applicable. The dual culture assay provides a standardized strategy for studying bacteria–fungi interactions, which can easily be adapted to various projects interested in studying such interactions. Similarly, our secretion assay can be used to evaluate different secretion tags, and with minor modifications, it can be applied to other bacterial species beyond B. subtilis. These tools contribute to the wider synthetic biology community by enabling comparable and meaningful measurements across diverse projects.
We implemented well-considered controls for all experiments to validate our measurement accuracy and reliability. Control conditions were included to confirm assay performance, and key experiments were repeated to assess consistency and minimize variability. Through these validation steps, we ensured that our measurement data are both precise and trustworthy.