General Overview
Experiment 1: Biofilm Formation
Cycle 0
Overview
In the first experiment, we wanted to confirm whether the early biofilm formation was due to the presence of cereulide and to find the concentration of cereulide required for biofilm formation. Cereulide acts as a potassium ionophore that triggers activity of a kinase called KinC, which is involved in activating genes that cause biofilm formation[1]. For this experiment, we used valinomycin instead of cereulide to trigger potassium efflux to observe an early biofilm formation since they share similar chemical structures and they both have ability to cause potassium efflux in B. subtilis, which is the chassis we used for our project[1]. Furthermore, we could expect the similar trend of biofilm formation induced by cereulide against time after exposing valinomycin to B. subtilis for different periods of time.
Key Achievements
- To test whether valinomycin causes early biofilm formation in B. subtilis.
- To access at what concentration valinomycin could trigger the biofilm formation in B. subtilis.
96-wells plate design
Each well contained 200 µL of solution (198 µL B. subtilis in LB or TSB medium + 2 µL valinomycin). There are two plates for each experiment, one plate with KCl and without KCl. We added a plate containing KCl to examine the effect of KCl in inhibiting K+ efflux in B. subtilis cells. [3]
Biofilm staining
After incubation, B. subtilis in the medium was washed out. The remaining biofilm on the wall of each well was stained with 0.1% (v/v) crystal violet solution diluted in water, and the biofilm amount was quantified by measuring the absorbance (570 nm) of crystal violet using a plate reader.
Figure 1: Dilution Assay [Biofilm formation intensity of Bacillus subtilis suspended in LB medium against different concentrations of Valinomycin (0 µM, 1.5 µM, 3 µM, 6 µM, 10 µM)]
Figure 2: Dilution Assay (With KCl added) [Biofilm formation intensity of Bacillus subtilis suspended in LB medium with 150mM KCl against different concentrations of Valinomycin (0 µM, 1.5 µM, 3 µM, 6 µM, 10 µM)]
Figure 3: Time-sensitive Assay [Biofilm formation intensity of Bacillus subtilis suspended in LB medium with different concentrations of Valinomycin added (0 µM, 1.5 µM, 3 µM, 6 µM, 10 µM) against incubation time (43-48 hrs)]
Figure 4: Time-sensitive Assay (With KCl added) [Biofilm formation intensity of Bacillus subtilis suspended in LB medium with 150 mM KCl with different concentrations of Valinomycin added (0 µM, 1.5 µM, 3 µM, 6 µM, 10 µM) against incubation time (43-48 hrs) ]
Conclusion
The above evidence shows that valinomycin can induce a higher rate of biofilm formation of Bacillus subtilis, and that the concentration of valinomycin has a positive correlation with the intensity of biofilm formation of Bacillus subtilis. It also suggests that Valinomycin would in fact trigger K+ efflux, as our results show that there are no distinct trends that appear from our experiments which contain potassium chloride [Dilution trials 1 and 2 (KCl) and Time-sensitive trials 1 and 2 (KCl)] . From previous studies, it is known that cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties[1], [2]. Therefore, it is very likely that biofilm formation in B. subtilis is proportional to cereulide concentration.
Experiment 2: Potassium Efflux
Cycle 0
Overview
Since our biosensor is based on the potassium ion efflux effect of B. subtilis caused by cereulide, it is important to know how cereulide concentration affects the potassium ion efflux rate (Yang et al., 2023). Thus, the main goal of this experiment is to quantify the potassium ion efflux effect on B. subtilis caused by valinomycin, a potassium ionophore that is highly similar to cereulide. To do this, a potassium biosensor (BBa_K1682009) developed by the 2015 iGEM HKUST-RICE team was used to measure extracellular potassium levels under the effect of valinomycin (iGEM15_HKUST-Rice, 2015) (Figure 1).
Key Achievements
- To test for the hypothesis of valinomycin-induced potassium efflux.
- To quantify the effect of valinomycin concentration on potassium ion efflux rate in B. subtilis.
Establishing a Standard Curve for Extracellular Potassium Levels
The HKUST-RICE team's genetic circuit allows the detection of various concentrations of extracellular potassium by lowering the expression of GFP as the potassium concentration increases. For a fair comparison, we first exposed the genetically modified E. coli (BBa_K1681009) to various known extracellular potassium concentrations and compared the resulting GFP signals to set up a standard curve for fluorescence levels in various potassium concentrations.
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Preparation of Known Potassium Concentrations
Various known potassium concentration solutions were prepared based on the protocol written by the HKUST-RICE team. Furthermore, additional concentrations that were not included in the original protocol were also tested.
Different concentrations of known potassium concentratnion medium:
0.01, 0.02, 0.05, 0.1, 0.2, 0.5mM standard K medium wass prepared by 1:9 dilution with K0 medium.
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Sample Preparation
The genetically modified E. coli from the HKUST-RICE team was washed with a 0.8% NaCl solution. After washing, 25 μl of the genetically modified E. coli was added to various known potassium concentrations. The bacterial culture was then incubated for 5 hours and measured by the FlexStation Multi-mode Microplate Reader.
Investigation of Valinomycin-Induced Potassium Ion Efflux in B. subtilis
Solutions with different concentrations of valinomycin were used to study the potassium ion efflux effect of valinomycin on B. subtilis. Moreover, we evaluated the extracellular potassium levels at different time points during the experiment.
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Preparation of valinomycin with different concentrations
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Sample Preparation
After resuspending the B. subtilis culture with different valinomycin concentrations, the samples will be incubated for the respective time periods. After centrifugation, the supernatant of each sample will be collected. The supernatant will then be mixed with the potassium biosensor (Bba_K1682009). After 5 hours of incubation, the fluorescence levels of the resulting samples will be measured. The data will then be compared with the standard curve to identify the extracellular potassium levels.
Results
By assuming the initial extracellular K+ concentration is 0mM, Fig1.2c.3 indicates that the potassium ion efflux rate is proportional to valinomycin concentration with an increasing trend. Since the GFP production is low at high extracellular K concentration and the little variation of the biosensor would significantly affect the overall results, the fluctuations of extracellular potassium ion concentrations after 4 minutes might be attributed to the lower sensitivity of the potassium ion biosensor (BBa_K1682009) at higher potassium ion concentrations. The extremely large value at 6 minutes with 6uM of valinomycin concentration is omitted. One plausible explanation for the excessively high value could be contamination by potassium ions. .
Conclusion
The above evidence shows that valinomycin can cause potassium ion efflux in B. subtilis, and the potassium ion efflux rate is proportional to valinomycin concentration. From previous studies, it is known that cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties [3] Therefore, it is safe to assume that potassium ion efflux in B. subtilis is proportional to cereulide concentration.
Experiment 3: Fluorescence Assay on Circuit
Cycle 0
Overview
This study focuses on the experimental testing of a genetic circuit to detect cereulide level. Cereulide is a potassium ionophore that disrupts potassium ion gradients, and we have leveraged this property to create a biosensor. The genetic circuit utilizes the endogenous potassium-sensing pathway in Bacillus subtilis to detect cereulide-induced potassium ion efflux and convert it into a measurable signal, red fluorescence.
Key Achievements
The experiment aims to assess the functionality and performance of the genetic circuit (Cycle 0) designed for detecting cereulide levels in food samples. Specifically, the experiment aims to:
- Determine the responsiveness of the genetic circuit to valinomycin, a structurally similar ionophore to cereulide, by measuring the circuit's output (RFP fluorescence) at various valinomycin concentrations at various time points.
- Validate whether valinomycin can be used as a substitute for cereulide in triggering the genetic circuit, confirming that the responses to cereulide and valinomycin are similar.
- Establish the lower detection limit of the circuit, determining the minimum concentration of valinomycin (or cereulide) required to activate the circuit and produce a measurable signal.
- Measure the time it takes for the circuit to trigger and produce the desired output, providing information about the responsiveness and kinetics of the system.
- Collect data to evaluate the suitability of the genetic circuit for practical use in detecting cereulide in food samples.
The experiment aims to provide valuable insights into the circuit's sensitivity, specificity, and response dynamics, which are essential for its potential application as a biosensor for cereulide detection in real-world scenarios.
The experimental design for measuring the effect of valinomycin on
genetically engineered B. subtilis (Cycle 0) consists of several key
steps and components:
Materials:
- Genetically engineered B. subtilis
- Brain Heart Infusion (pH 8.5, 5 μg/ml CHL)
- DMSO
- Valinomycin (12.5 mg/mL)
- Autoclaved PBS (pH 7.4)
- 96-well plate (black, clear flat bottom)
Experimental Workflow:
Day 0: inoculation
1. Inoculate genetically engineered B. subtilis in 5 mL of Brain Heart Infusion (pH 8.5, with 5 μg/ml chloramphenicol) and allow it to grow overnight. This step provides a culture of B. subtilis (cycle 0) for the experiment.
Day 1: Experimental Setup
1. Prepare a 1.25 mg/mL valinomycin solution. To do this, combine 30 μL of 12.5 mg/mL valinomycin with 270 μL of DMSO and mix thoroughly. This solution will serve as the stock solution for valinomycin.
2. Prepare various valinomycin concentrations according to the required table. Combine Brain Heart Infusion (pH 8.5, with 5 μg/ml chloramphenicol), the stock valinomycin solution, and DMSO as specified to create mixtures of different valinomycin concentrations.
3. Store each of the 20 mL mixtures in separate 50 mL Falcon tubes. Incubate these tubes in a shaking incubator at 37°C and 300 rpm. This step will allow the B. subtilis culture to be exposed to different valinomycin concentrations.
Sample collection
4. Remove one Falcon tube from the shaking incubator. For each
concentration being tested, transfer 1.5 mL of the mixture into a 1.5 mL
Eppendorf tube.
5. Centrifuge the Eppendorf tubes for 3 minutes at 14,000 rpm to pellet
the cells. Discard the supernatant.
6. Add 1 mL of PBS (pH 7.4) to each tube and resuspend the cell pellet
by pipetting up and down. Centrifuge again for 3 minutes at 14,000 rpm
and discard the supernatant.
7. Repeat step 6 (resuspension in PBS and centrifugation) two more
times.
8. After the final PBS resuspension, store the Eppendorf tubes on ice or
at 4°C.
9. Repeat steps 5 to 8 for each Eppendorf tube every 30 minutes for a
total of 8 hours, starting from the 0-hour time point. This allows for
multiple data points over time.
Data Collection
10. After 8 hours of exposure to valinomycin, pipette 200 μL from each
Eppendorf tube (technical replicates) into a 96-well plate.
11. Measure the optical density at 600 nm (OD600) to determine cell
growth and RFP (red fluorescent protein) fluorescence using the
appropriate excitation (ex: 584 nm) and emission (em: 607 nm)
wavelengths. These measurements will provide data on how the genetic
circuit responds to different valinomycin concentrations over time.
This experimental design allows a systematic assessment of the genetic circuit's response to valinomycin/cereulide and evaluates its sensitivity and dynamic characteristics. By collecting data at various time points and concentrations, conclusions about the circuit's performance can be drawn.
Figure 1: RFP fluorescence on cycle 0 upon exposure to 3-9 μM valinomycin, fold change when compared to no valinomycin added, em:584 nm ex:607 nm
In our first trial, we tested the circuit with 3 μM, 6 μM, and 9 μM of valinomycin. All three concentrations triggered RFP production at around 4 hours and showed a 60-fold increase in RFP fluorescence after 6 hours when compared to no valinomycin added.
After our first trial, we wanted to find out the lower detection limit of our circuit, so we tested the circuit with 0.03 μM, 0.3 μM, 3 μM, and 4.5 μM of valinomycin in our second trial.
RFP fluorescence on cycle 0 upon exposure to 0.03-4.5 μM valinomycin, fold change when compared to no valinomycin added, em:584 nm ex:607 nm
In our second trial, concentrations of 0.3 μM above triggered RFP production at around 4 hours and showed a 20-fold increase in RFP fluorescence after 6 hours when compared to no valinomycin added. At the same time, we found out that 0.03 μM of valinomycin failed to trigger the cycle 0 circuit.
Due to a limited stock of cereulide, we previously used valinomycin to test our cycle 0 circuit and assumed that valinomycin and cereulide would cause the same effect on our circuit as they have very similar structures and effects on gram-positive cells5. To validate our assumption, in the third trial, 0.3 μM of cereulide and valinomycin were used, respectively, to test our circuit. The intervals of sample collection are also shortened to 30 minutes to get more comprehensive data.
RFP fluorescence on cycle 0 upon exposure to 0.3 μM valinomycin and cereulide, fold change when compared to no valinomycin added, em:584 nm ex:607 nm
The results of the third trial showed that the same concentration of cereulide and valinomycin cause very similar effects on our cycle 0 circuit, and therefore our assumption is validated.
Cycle 1
Overview
In this study, we designed and implemented a genetic circuit to detect the presence of cereulide using genetically engineered B. subtilis. Building on our previous cycle 0, we created Cycle 1 of the genetic circuit, which includes a cereulide-activated and cereulide-repressed component.
The purpose of this experiment is to determine how different concentrations of valinomycin, a cereulide analog, affect our genetically engineered B. subtilis which contains our Cycle 1 genetic circuit. To indicate the absence of cereulide, a green fluorescence protein (GFP) is produced in the genetic circuit whereas a red fluorescence signal (RFP) is generated when exposed to cereulide.
At set intervals throughout the experiment, we take samples to analyze the optical density (OD600), red fluorescent protein (RFP) fluorescence, and green fluorescent protein (GFP) fluorescence. The results can shed light on the genetic circuit's reaction to valinomycin.
Key Achievements
The primary aim of this experiment is to assess how a genetically modified B. subtilis strain with a Cycle 1 genetic circuit responds to different concentrations of valinomycin, a compound that's analogous to cereulide. The broader goals include confirming the functionality of the genetic circuit in generating a distinct response, specifically an increase in red fluorescence (RFP) when exposed to valinomycin, and characterizing its sensitivity.
The aim is to provide insights into the circuit's performance by collecting data on optical density (OD600), RFP fluorescence, and green fluorescent protein (GFP) fluorescence over a 5-hour period.
Furthermore, this experiment is an important step in the development of a biosensor for the detection of cereulide in food samples. The findings of this study will assist and inform future improvements in circuit design and optimization for increased sensitivity and dependability.
The experimental design for this study involves exposing a genetically engineered B. subtilis strain carrying a Cycle 1 genetic circuit to different concentrations of valinomycin, a cereulide analog, and monitoring the response of the circuit over an 5-hour period. The key components of the experimental design are as follows:
Material and Chemicals:
- Brain Heart Infusion (pH 8.5, 5 μg/ml CHL)
- DMSO
- Valinomycin (12.5mg/mL)
- Genetically engineered B. subtilis
- Autoclaved PBS (pH 7.4)
- 96-well plate (black, clear flat bottom)
Experimental Workflow
Day 0: Inoculation
1. Genetically engineered B. subtilis is inoculated in 5 mL Brain Heart Infusion (pH 8.5, 5 μg/ml CHL) and allowed to grow overnight.
Day 1: Valinomycin Exposure
1. Prepare a 1.25 mg/mL valinomycin solution using DMSO.
2. Prepare different valinomycin concentrations (0 μM, 0.3 μM, 3 μM, and 6 μM) by mixing valinomycin with Brain Heart Infusion and DMSO.
3. Incubate the mixtures with genetically engineered B. subtilis for 8 hours at 37°C, 300rpm in shaking incubator.
Sample Collection and Processing
4. Take each falcon tube from the shaking incubator. Pipette 1.5mL of the mixture into 1.5 mL Eppendorf tube for each concentration tested and centrifuge for 3 minutes at 14000 rpm. Discard the supernatant.
5. Add 1 mL of PBS and resuspend the pallet by pipetting up and down. Centrifuge for 3 minutes at 14000 rpm. Discard the supernatant.
6. Repeat step 5 for 2 more times.
7. Add 1 mL of PBS and resuspend the pallet by pipetting up and down. Store the tube on ice or at 4 degrees Celsius 3. Incubate the mixtures with genetically engineered B. subtilis for 8 hours at 37°C, 300rpm in shaking incubator.
8. Repeat step 5-8 every 60 minutes for 5 hours, starting from 0-hour time point.
Data Collection:
9. After the 8-hour incubation period, transfer 200 μL of each resuspended sample (in triplicate) into a 96-well plate.
10. Measure optical density (OD600), red fluorescence (RFP) fluorescence (ex: 584 nm em: 607 nm), and green fluorescence protein (GFP) fluorescence (ex: 504 nm em: 515 nm) to assess the circuit's response to different valinomycin concentrations.
This experimental design allows for the systematic assessment of how the genetic circuit of cycle 1 responds to valinomycin at various concentrations, helping to characterize its sensitivity, dynamic range, and kinetics over time. The collected data provides insights into the performance of the genetic circuit and serves as a foundation for circuit optimization, dry lab modeling, and further development.
RFP fluorescence on cycle 1 upon exposure to 0.3-6 μM valinomycin, Fluorescence/OD, em:584 nm ex:607 nm
RFP fluorescence on cycle 1 upon exposure to 0.3-6 μM valinomycin, fold change when compared to no valinomycin added, em:584 nm ex:607 nm
Upon the addition of valinomycin, there was a significant 40-120 fold increase in RFP at around 4 hours when compared with the 0 μM solvent control the result is consistent with cycle results.
GFP fluorescence on cycle 1 upon exposure to 0.3-6 μM valinomycin, fluorescence/OD , em:504 nm ex:515 nm
For the GFP, it is found that the production of GFP is longer than anticipated and the degradation of GFP also takes a considerable amount of time, therefore, further tuning of the circuit is needed.
Experiment 4: Characterization of Degradation Tags using Fluorescence Assay
Overview
It is crucial to test out every construct to understand the stability and degradation kinetics among all the degradation tags, as well as providing data for the bioinformatic database. Fluorescent assay is one way to investigate the properties of the degradation tags. In addition, fluorescent microscopy and western blotting could also be performed to achieve even more accurate results.
Key Achievements
In order to XXX, we need YYY
Fluorescent Assay
Measuring fluorescence using a microplate reader allows for high throughput analysis for the degradation tags to be tested. Each construct is back diluted to fresh LB broth with appropriate antibiotics. Any GFP production is monitored and measured every 1 hour by measuring OD600 and fluorescence (Ex:504 nm; Em:515 nm) until at least a 5-hour time-point is reached.
To study the kinetics on whether the degradation tag functions conditionally to degrade GFP when adapter protein SspB from E. coli is expressed, our construct allows the expression of SspB only upon the addition of Isopropyl ß-D-1-thiogalactopyranoside (IPTG) which allow us to first select ‘stable’ degradation tags before testing if they are SspB dependent. For any ‘stable’ degradation tag constructs that have reached a 5-hour time-point, IPTG is added and the GFP stability is closely monitored every 5 minutes as previously described.
Fluorescent Microscopy and Western Blotting
Fluorescent microscope allows visual monitoring of the degradation kinetics among individual bacteria cells in real time and western blotting allows for specific detection plus quantification of GFP at different time-points. This is essential as combining all methods together reduces false-negative results. Therefore, other than expressing a green fluorescent protein with a degradation tag of our choice, we also fused a 6xHistag to our GFP construct for western blot analysis.
Same as the fluorescent assay using the microplate reader, live cell imaging is taken every hour until at least a 5-hour time point is reached. Then select the 'stable' degradation tag for degradation analysis by adding IPTG and image every 5 mins for at least 30 mins. As for western blot, cells are harvested when OD600~0.2 and another half of cells are harvested after induction of IPTG for 1 hour. The accumulation or degradation of GFP can be determined by western blot using anti-His tag antibody.
For future work, let's go back to: Degradation Tag Library: Future Work