Overall Background

This year, our team focused on engineering Synechocystis sp. through adaptive laboratory evolution (ALE) to develop a strain capable of thriving under high pH conditions. We employed a fluorescence-based quantification assay to measure the polyhydroxybutyrate (PHB) content in Synechocystis, evaluating how elevated pH levels influence PHB production. Additionally, we utilized a PrestoBlue assay to assess the cytotoxicity of PHB extracted from the evolved cells on the Detroit 551 cell line, determining its impact on cell viability.

• Fluorescence-based quantification assay for PHB content in Synechocystis: Analyzing the effect of high pH on producing PHB contents

• Prestoblue assay (Cytotoxicity assay): Assessing the effect of extracted PHB on Detroit 551 cell line viability.

Fluorescence-based quantification assay for PHB content and Prestoblue assay were critical to the ‘test’ phase of our Engineering Cycle. However, both measurement needs the normalization reference (standard curve) to quantify the PHB produced from Synechocystis. Also, prestoblue requires a standard curve to quantify the metabolic activity of the living cells. Accordingly, our objectives for this year’s measurement protocols were the following:

Measurement Part 1: Fluorescence-based quantification assay for PHB content in Synechocystis

Measurement Background

The fluorescence-based quantification assay is a widely used method for determining the polyhydroxybutyrate (PHB) content in Synechocystis sp., particularly when studying the effects of environmental stressors like pH on metabolic pathways. PHB, a biodegradable polymer, is synthesized by many microorganisms as an energy-storage molecule under nutrient-limited conditions. In this assay, the PHB molecules within the Synechocystis cells are stained with a fluorescent dye, such as Nile Red, which specifically binds to hydrophobic regions. Upon excitation at specific wavelengths, the intensity of fluorescence emitted correlates with the amount of PHB present. By measuring fluorescence, researchers can quantitatively assess how different pH conditions influence PHB production in Synechocystis, offering insights into the potential for optimizing bio-plastic production in environmentally stressed cyanobacteria. This approach provides a sensitive, rapid, and non-destructive means to monitor PHB levels, facilitating the study of metabolic adaptations in engineered strains.

Measurement Principle

The principle of the fluorescence-based quantification assay for PHB content in Synechocystis relies on the interaction of a hydrophobic fluorescent dye, such as Nile Red, with intracellular PHB granules. Nile Red selectively binds to the hydrophobic regions of PHB within the cells, and when excited by a specific wavelength of light (usually in the range of 488-550 nm), it emits fluorescence in the red spectrum. The intensity of this fluorescence is directly proportional to the amount of PHB present in the sample. By measuring the fluorescence intensity using a spectrofluorometer, researchers can accurately quantify the PHB content. This method allows for rapid, non-destructive, and highly sensitive detection of PHB, providing valuable insights into the metabolic responses of Synechocystis under varying environmental conditions, such as high pH stress.

Measurement Protocols

Synechocystis sp. cultures grown under different pH conditions (7.5, 9.5, 11.0, and 11.5)

Nile Red dye

Chloroform-methanol solution (for PHB extraction)

Fluorescence microscope

Spectrofluorometer

Phosphate-buffered saline (PBS)

Microcentrifuge tubes

Pipettes and sterile tips

Scale for dry weight measurement

50 µm scale bar for imaging

Procedures

Sample Preparation

Grow Synechocystis sp. cultures under varying pH conditions (7.5 to 11.5) to induce PHB production.
Harvest cells by centrifugation at 5000 x g for 10 minutes and wash the pellet twice with phosphate-buffered saline (PBS).
Resuspend the cell pellets in PBS for further analysis.

Nile Red Staining and Fluorescence Measurement

Prepare a 1 mg/mL Nile Red stock solution in acetone.
Add 10 µL of Nile Red solution to each Synechocystis cell suspension, ensuring thorough mixing to stain the intracellular PHB granules. The number of cells must be normalized by OD750.
Incubate the stained cells in the dark for 10 minutes at room temperature.
Measure the fluorescence intensity using a spectrofluorometer, with excitation at 488 nm and emission at 590 nm. Record the fluorescence values for each sample.
Plot standard curve of fluorescence intensity against the PHB content obtained from the chloroform-methanol extraction method to assess the linear correlation.

A total of 17 samples were prepared to plot the standard curve. Here are the standard curve sample measurements:

Fluorescence Microscopy Imaging

Prepare glass slides with a small aliquot of Nile Red-stained Synechocystis cells for imaging.
Capture fluorescence images using a fluorescence microscope, with the appropriate filters for Nile Red detection (excitation at 488 nm and emission at 590 nm).
Compare fluorescence intensity and distribution of PHB granules in cells grown under different pH conditions. Use a 50 µm scale bar for image calibration.

 PHB Extraction and Quantification

After fluorescence measurement, proceed with the chloroform-methanol extraction method to isolate PHB from the cells.
Normalize PHB content to cell dry weight and plot the values against fluorescence intensities.
Generate a scatter plot of fluorescence intensity versus PHB content and draw a linear regression line. Calculate the R² value to confirm the correlation between fluorescence intensity and intracellular PHB content.

Plot a standard curve using a spreadsheet to show the known concentrations of the PHB standard and their corresponding fluorescence values.

Using the standard curve, find the corresponding PHB concentration for each sample based on their fluorescence values.

0.1219 X Fluorescence intensity (AU) + 3.546 = PHB content (wt%)

This equation can be used to calculate the PHB content (wt%) by measuring the fluorescence intensity (AU) measured by microfluorometer.

The outcomes of this quantification provided the following PHB contents for the respective pH growth conditions:

Measurement Discussion

In fluorescence-based quantification assays for PHB content, normalizing the number of Synechocystis cells is critical to ensure accurate and reliable measurements. Since fluorescence intensity correlates with PHB content, it is essential to control for variations in cell concentration between samples, as higher cell numbers could artificially increase fluorescence intensity, leading to overestimation of PHB levels. Normalization of the cell count, typically achieved by adjusting the optical density (OD) at 750 nm, ensures that fluorescence differences are attributed to variations in PHB content rather than disparities in cell number. Without proper normalization, the fluorescence signal may reflect differences in biomass rather than true variations in PHB production, potentially skewing the results and limiting the ability to make accurate comparisons across different pH conditions. Therefore, accurate normalization is a fundamental step for ensuring that the fluorescence intensity accurately reflects intracellular PHB content.

Measuring fluorescence in control samples is crucial for accurately calculating the standard curve due to the presence of background fluorescence. Even in the absence of the target compound, such as PHB, background fluorescence from the cells, medium, or assay components can contribute to the overall signal. Without accounting for this, the measured fluorescence values may be artificially elevated, leading to inaccurate quantification of the target molecule. By including a control sample without PHB, we recommend that future iGEMers can subtract the background fluorescence from the experimental values, ensuring that the standard curve reflects only the specific fluorescence from PHB. This correction enhances the precision of the standard curve and ensures reliable measurements of PHB content across different samples.

Measurement Part 2: Prestoblue assay

Measurement background

The PrestoBlue assay, a widely used and versatile technique in cell biology, is an essential tool for accurately assessing cell proliferation and viability. This assay measures the metabolic activity of cells, providing a reliable and efficient method for estimating cell numbers and overall cell health.

Measurement Principle

The PrestoBlue assay operates on the principle of measuring cellular metabolic activity as an indicator of cell proliferation. It uses the non-toxic, colorless dye resazurin, which is readily absorbed by living cells. Once inside, resazurin is reduced by mitochondrial enzymes in metabolically active cells, converting it into resorufin, a highly fluorescent and colorful compound. The resulting fluorescence or absorbance intensity is directly proportional to the number of metabolically active cells in the culture. By quantifying the rate of resazurin's conversion to resorufin, the assay provides a reliable measure of cell proliferation, reflecting changes in cell numbers over time.

Materials

Detroit 551 cultured cells

PrestoBlue reagent (commercially available)

96-well microplate

Microplate reader

Pipettes and tips

Sterile cell culture hood or laminar flow hood

CO₂ Incubator

Extracted PHB

Procedures

Step 1: Cell Seeding

To prepare the cell samples, 96-well plates were used, and 1x10³ Detroit 551 cells were added to each well for the experiment. Additionally, standard curve cells were prepared by varying the number of cells to 0, 1x10³, 2x10³, 5x10³, 10x10³, and 15x10³. All samples were prepared for the duplicate measurements.

Step 2: Preparation of Test Wells and PrestoBlue Treatment

Aspirate the culture medium from the wells containing Detroit 551 cells.

Rinse the cells once with phosphate-buffered saline to remove any residual medium.

Add the 90 µL of cells containing 0, 2, 4, 6, 8, and 10% of PHB on each appropriate position of the 96 well plate.

Incubate the cells for 0, 24, 48, 72, and 96 hours of the treatment.

For each time point, PrestoBlue solution was added to the sample (10 µL) to each well-containing cell (final concentration should not exceed 10%).

Incubate the cells with the PrestoBlue reagent at 37°C for 30 minutes.

Step 3: Quantification of Cell Proliferation

Following incubation, a microplate reader was used to measure the absorbance at 570 nm (with a reference wavelength of 600 nm). Record the absorbance values for each well.

Calculate cell viability by comparing the absorbance values of the treated cells with those of the untreated control cells.

The PrestoBlue assay demonstrated a strong correlation (R²=0.991) between cell number and absorbance values, confirming its accuracy and consistency in measuring live cell numbers (Figure 4). This high correlation validates the PrestoBlue assay as a reliable method for quantifying changes in cell proliferation. Additionally, it highlights the success of our optimized experimental conditions. The standard curve shows that absorbance values up to 3 maintain a linear relationship with cell number, indicating that absorbance measurements should remain within this range for accurate quantification of live cells.

It is important to note that the absorbance values range from 0 to a maximum of 0.35, which falls within the linear range of the standard curve shown in Figure 4. This confirms the accuracy of using absorbance values to represent the number of live cells. Furthermore, the number of live cells can be accurately calculated from the absorbance measurement using the following equation:

(Absorbance 570/600 nm - 0.2938)/0.1745 = PHB content (wt%)

Measurement Discussion

The results of the PrestoBlue assay confirmed its effectiveness as a reliable method for assessing cell viability and proliferation by measuring the metabolic activity of Detroit 551 cells. A strong correlation (R²=0.991) between cell number and absorbance values was observed, indicating the assay’s accuracy and consistency in quantifying live cell numbers (Figure 4). This correlation validated our experimental conditions and demonstrated the linear relationship between absorbance and cell number, as long as absorbance values remained within the range of 0 to 3. Additionally, the effect of varying PHB concentrations (0 to 10%) on cell viability was measured over 96 hours (Figure 5). Absorbance values, which peaked at 0.35, remained within the linear range of the standard curve, allowing for an accurate representation of live cell numbers. Statistical analysis using a one-way ANOVA and Tukey's post hoc test further supported the significance of these findings. Cell viability could be accurately calculated using the equation provided, ensuring precise measurement of PHB's impact on cell health.

To obtain an accurate regression line, we included a negative control—specifically, a well with 0 cells. The x-axis represented the number of cells (×10³), and the y-axis displayed absorbance in arbitrary units (a.u.). Both the explanatory and response variables were carefully measured, resulting in a least-squares regression line with a strong positive linear correlation. We highly recommend future iGEM teams implement such control and calibration measures to ensure precise data collection. Using this regression line, we converted the absorbance values of each sample to the corresponding number of living cells, allowing us to accurately track cell proliferation at various time intervals.

This measurement technique will be valuable for future iGEM teams aiming to quantify cell viability and proliferation at different time intervals, as well as for those studying polyhydroxybutyrate (PHB) production, a popular subject in many iGEM projects focused on bioplastics and sustainability. Accurate assessment of cell health is critical for biomanufacturing in synthetic biology, where controlling the rate of cell proliferation and optimizing PHB production are essential for improving yield and system performance. To achieve our project goals this year, we developed a reliable protocol using the PrestoBlue assay to measure cell viability and incorporated methods to assess PHB content. By establishing a standard regression line and performing quantitative calculations, we were able to determine cell proliferation rates and correlate these with PHB production. Combining the PrestoBlue assay with quantitative statistics allows for robust, reproducible results that can guide PHB optimization. However, it is important to note that errors in measuring low cell counts could lead to negative or inaccurate readings, and special care should be taken to avoid errors when handling low-proliferation or low PHB-yielding samples.