We have engineered a hydrogel-based whole cell biosensor by utilizing the LasI-LasR quorum sensing system originally found in P. aeruginosa. This whole cell biosensor contains a LacZ gene as the reporter gene, encoding β-galactosidase. The detection and quantification of blue β-galactosidase activity after incubation in an 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal) solution act as a visible readout for the amount of quorum sensing signalling molecules, N-acylhomoserine lactones (AHLs) which ultimately used for rapid, sensitive and quantitative detection of microplastics. The insoluble nature of blue products makes the measurement unsuitable for spectrophotometric absorbance quantification. Therefore, the use of X-gal assay is restricted to solid-support approach such as colony filter lift assay which is a qualitative assay in which the individual coloration of colonies is assessed visually. During the process of research, we have developed and optimized a protocol of a quantitative assay for measuring β-galactosidase activity to evaluate the E. coli biosensing system on the hydrogel-based assay. We have performed several experiments to optimize the assay for rapid and consistency in sample preparation and quantification. Various aspects including hydrogel percentage, cell density at starting point, X-gal concentration, AHL induction time and the best time for result observation are optimized. Moreover, we modified a protocol from Trimborn et al., 2022 (1) to integrate the use of ImageJ-based ReadPlate plugin for quick and accurate measurement. The optimized quantitative X-gal assay based on hydrogel developed in this protocol provides an easy and rapid way of detecting and quantifying AHLs. Besides, the assay is standardized for reliability and reproducibility. Moreover, it can also be used in a standard laboratory without the need for any specialized equipment. We believe that this protocol contributes to future iGEM teams to quantitatively measure the β-galactosidase activity on solid supports such as agar plate, hydrogel or paper strip in an easy and rapid way.
Optimized quantitative X-gal assay
1. The design of the whole-cell biosensor
The engineered biosensor consists of two modules, the sensing module and the reporting module. The sensing module expresses the transcription factor LasR under the control of the constitutive T7 promoter in the plasmid pSB1C3. LasR can then bind to the AHL molecule, N-3-oxododecanoyl-homoserine lactone (3OC12-HSL). The formation of the LasR-AHL complex subsequently bind to an inducible promoter, pLasRL which activates the transcription of the reporting module. A LacZ gene is used as the reporting module, in which the development of the blue colour would be induced by the LasRL-AHL complex after the addition of X-gal. The resulting plasmid was named pSB1C3-LasR-pLasRL-LacZ. The plasmid was transformed into the E. coli BL21 strain.
2. Characterization of the biosensor with the LacZ reporting module
A single colony of the biosensor were inoculated into 1 mL of LB broth with 34 mg/mL chloramphenicol (LCM). It is incubated at 37°C, 230 rpm overnight. For bacterial subculture, the starter culture was added to LCM in the ratio of 1:99 starter culture to LCM in a 50 mL falcons. The falcon is put to incubate at 33°C, 220 rpm. It is allowed to grow for 3 hours until the bacterial concentration reaches OD600 of 0.3 and 0.7. 2 mL of the culture was transferred into a 15 mL culture tubes for induction with synthetic AHL, 3OC12-HSL at 1.0 X 10-6 M for finding the optimized AHL induction time and at seven concentration (1.0 X 10-5 M, 1.0 X 10-6 M, 1.0 X 10-7 M, 1.0 X 10-8 M, 1.0 X 10-9 M, 1.0 X 10-10 M and 1.0 X 10-11 M) for sensitivity test. All the tubes were incubated at 37°C and 220 rpm, and 300 μL of the cultures were withdrawn at intervals of 2 h for a total experimental time of 6 h. The cells were collected by centrifugation, washed twice with phosphate buffered saline (PBS) buffer and resuspended in 200 μL of 2% and 3% of hydrogel in a microplate.
3. Quantification of β-galactosidase activity
After cell resuspension using hydrogel, 5 mg/mL and 10 mg/mL of X-gal substrate solution were added into wells of the 96-well plate with biosensor cells. The plates were then incubated at room temperature to allow blue color development, which can be quantified by taking photographs at 5 minutes interval using smartphone camera and then analyzing the images with an ImageJ-based plugin.
4. Set up for image taking
Any reflection or shadows in the pictures would affect the quantification using ImageJ. Thus, we designed a set up to take pictures to prevent any reflection. All pictures are taken from the bottom of the plate for a more evenly distributed colour result.
Fig 1. Full set up to take pictures.
Fig 2. Set up for smartphone photo taking.
5. Analyzing the photographs using ImageJ and ReadPlate 3.0
Download ImageJ from https://imagej.net/ij/download.html and Readplate 3.0 from https://forum.image.sc/t/readplate-3-0-release/42008.
i. After downloading the images, set the contrast and saturation of the image to 100 to articulate subtle differences in color intensity.
ii. Open the ImageJ software. Go to “Analyze” → “Set Measurements”. Select “Area”, “Mean gray value”, “standard deviation”, “modal gray value” and “Min & max gray value”. Select the results to be 5 decimal places.. These are selected to improve the accuracy of quantifying the color intensity even if the color is unevenly distributed in a well.
iii. “File” → “Open” and choose the image from your computer. Ensure that the image is in the correct orientation.
iv. Select the rectangle. Drag the rectangle such that the vertices of the rectangle are on the center of the wells A1, A12, H1 and H12 (A 96 well plate is used in this report).
v. “Plugins” → “Macros” → “Run”
vi.From “Readplate_wr”, select “source code” → “Readplate 3.0”. Select 96 for the number of wells.
vii.Select “Red” for “Channel”:
viii.For the “parameters”, use the default settings
ix.Check whether the grid generated by the software fits each well. If the grid is at the center of each well, and the color intensity is evenly distributed in each well, click “OK”. Else, click “cancel” and increase the “Diameter of main circle (in pixels)” in Step 8
x.Click “OK” in “Checking Results”, then, select “Yes” in save results.
Name your file with the type of document as .csv or .xlsx to compile a spreadsheet.
6. Results
i. Hydrogel percentage
The whole-cell biosensor was immobilized in 2% and 3% hydrogel and added in a 96-well plate. The viscosity of hydrogel affects the performance of the biosensor, for example, if the hydrogel percentage is too high, the pellet would not be able to resuspend. On the other hand, if the hydrogel is too watery, it will easily evaporate.
Fig 3. Images of sample using 3% hydrogel to resuspend (left) and 2% hydrogel to resuspend (right).
The above result demonstrates that when the hydrogel percentage is too high (3%), suspension remains in the well which affects the performance and visibility of the biosensor. Hence, 2% hydrogel is chosen for the following investigation.
ii. Bacterial Optical Density
We studied the effect of induction cell density at induction time by comparing the β-galactosidase activity on two bacterial densities y at OD600 of 0.3 and OD600 of 0.7 for 3OC12-HSL induction.
Fig 4. β-galactosidase activity of biosensor on two different bacterial concentrations (OD600) for AHL induction. The biosensor was induced by 3OC12-HSL at 1.0 X 10-6 M for 0h and 2h, and the blue luminescence production rate is quantified as β-galactosidase activity. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments; error bars indicate the standard deviations.
The result shows that biosensor cells with OD600 of 0.3 has a higher β-galactosidase activity with more obvious difference between samples with 3OC12-HSL and without 3OC12-HSL induction. While in the OD600 of 0.77, sample have less difference with the control samples due to the rapid background expression of β-galactosidase in the absence of inducer, X-gal caused by the large cell number in the sample. Thus, OD600 0.3 is chosen for the following investigation to minimize the effect of background expression.
iii. Concentration of X-gal
The concentration of X-gal would affect the visibility of the blue colour formed. The intensity of the blue color with different concentrations of X-gal (5 mg/mL and 10 mg/mL) were compared to determine the most suitable X-gal concentration for the blue product observation and quantification.
Fig 5. Mean Auncorr (an “uncorrected value” as an output value from the Readplate 3.0 analysis which indicate the intensity of blue colour formed after X-gal induction) on different X-gal concentration. The biosensor was induced by 3OC12-HSL at 1.0 X 10-6 M for 2hr. Then, 5 mg/mL and 10 mg/mL of X-gal were added to the samples. Colour development was carried out for 15 minutes at room temperature. Samples without X-gal induction (0h) were used as negative controls. The blue colour of samples was compared to samples at 0h without AHL induction. The result are the means of triplicate experiments; error bars indicate the standard deviations.
The result shows that the X-gal with 10 mg/mL concentration has the highest mean Auncorr value which indicates the highest intensity of blue luminescence emission. Comparing with 5 mg/mL samples, X-gal with 10 mg/mL is more suitable to observe the β-galactosidase activity between samples with AHL and without AHL as it has a more obvious colour difference than that of 5 mg/mL X-gal without saturation. Therefore, 10 mg/mL X-gal is chosen for the subsequent investigation for a better visualization of the blue luminescence.
iv. AHL Induction Time
To evaluate the ability of the engineered biosensor to detect synthetic AHL compound 3OC12-HSL, the biosensor was induced by 1.0 X 10-6 M 3OC12-HSL for 0h, 2h, 4h and 6h. Meanwhile, bacterial optical density was recorded at every time point to study the growth of biosensor cells.
Fig 6. Optical density at 600 nm (OD600) at every two hours after AHL induction. The biosensor was induced in AHL at 1.0 X 10-6 M and incubated in shaker at 33°C, 220 rpm. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments.
A visible blue colour was developed in the biosensor spot after 2h, 4h and 6h of incubation with 1.0 X 10-6 M 3OC12-HSL using 5 mg/mL and 10 mg/mL X-gal. The negative control spot showed a yellow colour representing the uninduced biosensor cells.
Fig 7. 96-well plate photo of biosensor with different X-gal concentration every two hours after AHL induction. The biosensor was induced in AHL at 1.0 X 10-6 M (+ve) and incubated in shaker at 33°C, 220 rpm. The biosensor without AHL induction (-ve) was used as the negative control. Three X-gal concentrations, 0 mg/mL, 5 mg/mL and 10 mg/mL were added in samples. The result is a triplicate experiment. Image is taken 15 minutes after X-gal induction.
The result shows the obvious colour difference between 0h and 2h AHL incubation time, where 2h samples have relatively higher colour intensity than that of 0h. Although 4h and 6h samples have high colour intensity like the 2h samples, the colour difference between samples with and without AHL induction are smaller than that of 2h samples. Meanwhile, there were a gradual increase of OD600 from 0h to 4h, while the OD600 has a slight decline after 4h (Fig. 6). It indicated that the bacterial growth was inhibited as the incubation time increased. Therefore, we chose to incubate the samples 2 hours after AHL induction to minimize the effect caused by fluctuated bacterial density for a better visualization of the result.
v. Development Time
As the β-galactosidase requires time to produce blue coloured product after binding with X-gal, time of development is required to show an accurate result. To evaluate the suitable development time, the biosensor was induced in AHL at 1.0 X 10-5 M, 1.0 X 10-11 M for 2hr. Colour development were recorded over time after X-gal induction.
Fig 8. Linear regression of the colour development of biosensor over time towards 3 concentrations of AHL. The biosensor was induced in AHL at 1.0 X 10-5 M, 1.0 X 10-11 M for 2hr. Data is collected at 0min, 10min, 20min and 30min after X-gal induction. The colour intensity over time is quantified. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments; error bars indicate the standard deviations. R2 correlation coefficient.
The result suggested that the colour intensity of the blue coloured product increases with development time after X-gal induction where 20-30 minutes shows the best result. Hence, for the following investigation, 20-30 minutes are chosen for colour development after X-gal induction. Taken together, we have developed a protocol which allows for the fast and convenient detection of 3OC12-HSL after 2 hour induction time and visualization of the blue color development in 30 minutes.
vi.Sensitivity of the hydrogel-based Assay to detect AHL molecules
The sensitivity of the biosensor is characterized by quantifying the β-galactosidase activity in response to a range of concentrations of AHL.
Fig 9. β-galactosidase activity of biosensor towards eight concentrations of AHL. The biosensor was induced in AHL at 1.0 X 10-5 M to 1.0 X 10-11 M for 2hr, and the blue luminescence production rate is quantified as β-galactosidase activity. The biosensor without AHL induction was used as the negative control. The result are the means of triplicate experiments; error bars indicate the standard deviations. Result is taken 30 minutes after X-gal induction.
Fig 10. Image of biosensor activity towards eight AHL concentration. The biosensor was induced in AHL at 1.0 X 10-5 M (left) to 1.0 X 10-11 M (right) for 2hr. The biosensor without AHL induction(-ve) was used as the negative control. Image is taken 25 minutes after X-gal induction.
As shown in Fig. 9 and Fig. 10, the β-galactosidase activity is at a steady rate from 0 to 1.0 X 10-8 M 3OC12-HSL, and the rate started to increase when the concentration of 3OC12-HSL increased beyond 1.0 X 10-8 and peaked at 1.0 X 10-5, the highest concentration of 3OC12-HSL used. The result suggested that the hydrogel biosensor is able to detect AHL concentration as low as 1.0 X 10-7 M upon color development for 20 minutes. It is more sensitive when higher concentration of 3OC12-HSL is induced.
In summary, we have developed and optimized a protocol for quantitative X-gal assay based on 2% hydrogel which allows for the fast and convenient detection of 1.0 X 10-5 M to 1.0 X 10-11 M 3OC12-HSL using biosensor cells at OD600 of 0.3, 2h induction time and visualization of the blue colour development in 30 minutes using 10 mg/mL X-gal.
Reference
Trimborn, L., Hoecker, U., & Ponnu, J. (2022). A simple quantitative assay for measuring β-galactosidase activity using X-gal in yeast-based interaction analyses. Current Protocols, 2, e421.