Introduction
Before the successful engineering of biosensor cells, we conducted experiment 1.1 and
1.2 to test the stability of our chassis, Bacillus subtilis:
After successfully engineering the biosensor cells, we fine tune the protocol for biosensor hydrogel construction and conduct experiment 2 and 3 to test the performance of cells in the hydrogel matrix.
Protocols
Experiment 1.1: Sporulation and Germination
Overview
During the designing stage of PVA whole cell biosensor, we found that the sporulation of cells before embedding it into hydrogel could be an advancement we could apply to our whole cell biosensor. Sporulation, in other words, spores formation,occurs in nutrient-depleted environments, allowing bacteria to protect their DNA by encasing it in a tough, desiccated coating.When the environment is favo
Literature[2] has demonstrated that the shelf life of the whole-cell biosensor platform could be extended by the sporulation of B. subtilis. In the research, the hydrogel-embedded B. subtilis spores were subjected to temperatures ranging from -20 °C to 80 °C, covering the most common environmental conditions encountered in the transport of goods. It is proved that under all these extreme conditions, the performance of the sensor remains unperturbed after activation by nutrients.
Thereby, before we obtained gel construction material and engineered B. subtilis to be embedded in the gel, we had tried out the sporulation of B. subtilis according to the protocol provided by previous iGEM teams and research papers and found ways to optimize the sporulation process, simply put, adjust the protocol to induce B. subtilis cells sporulation within a shorter period.
Key Achievements
- Promote faster sporulation of B. subtilis
- Choose treatment to Inactivate the vegetative cells and obtain culture of spores only for embedding in gel
Section 1: Sporulation
OD600 before sporulation higher than protocol's with OD600 at
0.86/mL (protocol used 0.8/mL)
Inoculate in Freshly made 5 mL DSM(Check our protocol)
Result: 5 days to see >85% cells sampled become spores
Accidentally NOT follow Protocol's OD600 0.8/mL for sporulation:
We used OD600=0.69/mL (Lower) with the following settings:
- Inoculate in Freshly made 5 mL DSM
- Temperature: Room Temperature without putting it in shaking incubator
- Result(Figure 4): 36 hours can see >85% cells sampled become spores
Conclusion for Section 1
We had sorted modified B. subtilis sporulation protocol and reminders for upcoming iGEM teams that plan to do sporulation of B. subtilis. Cells with OD600 slightly lower than 0.8 at 0.7-0.75 is preferred to speed up the sporulation duration so that more cells could be sporulated in the DSM (Difco Sporulation Medium), by making cells as the “limiting reactants“ in this biological process.
Section 2: Isolation of spores from cells-spore mixture
As mentioned in the overview, spores are more robust and able to survive in harsh conditions, in order to find a way to kill the vegetative cells while keeping the spores able to germinate, the B. subtilis cells in LB and spores-cells in DSM are treated with the following conditions, followed by plate spreading on Tryptone Soy Agar with Chloramphenicol (CHL).
- 1. No treatment (Negative Control)
- 2. Exposure to UV light for 1.5hr
- 3. Place in heat block at 80°C for 1.5 hr
- 4. Place in heat block at 90°C for 1 hr
Conclusion for Section 2
We target the area where cells are all killed under harsh conditions while spores can be protected and germinate afterward when the condition is favorable for growth. From the plate incubated for 1.5 days(36 hours), we could see that exposure to heat block at 80°C for 1.5 hr could kill cells while not harsh enough to kill spores, thus yielding germinated cells on the plate. Therefore, after confirming spores do exist by observing stained green spores on glass slips through a microscope, we put the culture in a heat block at 80°C for 1.5 hr to confirm the mixture contains no vegetative cells. We further confirmed higher concentration of spores is obtained by comparing the cells seen in microscope before and after heating. (See Figure 6)
Experiment 1.2: Tolerance of Ethanol
Overview
From an interview with Professor Marshal Liu for test kit design consultation, he suggested using household solvent to extract cereulide from rice samples, for instance ethanol.
From literature[3], we found that Cereulide/Valinomycin Extraction from Food Sample by using methanol and ethanol has no significant difference.
Meanwhile, we know that ethanol at certain concentrations is used as a disinfection agent, which may kill our biosensor cells embedded in gel after the treated sample is applied to the gel disc. Therefore, we conduct experiments to find the optimal concentration of ethanol for cereulide extraction.
Key Achievements
- Make sure the B. subtilis biosensor cells embedded in gel would not be killed by the ethanol solution we applied for Cereulide Extraction.
Experimental Design
- Engineered Cycle 0 Cells with 1:10 and 1:100 dilution are centrifuged and resuspended in Milli-Q Water and spreaded 80 μL on Trypticase soy agar plate with Chloramphenicol added
- Engineered Cycle 0 Spores isolated in 80°C for 1.5 hr with 1:10 and 1:100 dilution are centrifuged and resuspended in Milli-Q Water and spreaded 80 μL on Trypticase soy agar plate with Chloramphenicol added
- 5 mL of ethanol with ascending concentration is poured respectively to the plates and leave for 15 mins (Pipette removal of 5 mL liquid)
- Incubate for 36 hours
Results
Colonies on plates on 0%,25% and 50% ethanol treated cells are not killed while those treated by 75 % ethanol solutions are mostly killed, and no viable colonies for 95% ethanol solution
Colonies on plates on 0%,25% ethanol treated spores are not killed while those treated by 50 % ,75 %,95% ethanol solutions are mostly killed and had no viable colonies for 95% ethanol solution
It could be concluded that the maximum concentration of ethanol solution we can use for cereulide extraction in rice is 50%.
Experiment 2: Fluorescence Protein viability in PVA Gel
Overview
To detect emetic toxin cereulide in food samples, our team aims to create a low-cost, user-friendly colorimetric biosensor that can detect high levels of cereulide on-site. Our bacteria circuit makes use of the potassium ion efflux effect caused by cereulide on B. subtilis, our chassis, red fluorescence signal would be expressed when cereulide exceeds the safe level. Subsequently, if the sample consists of a safe level of cereulide, green fluorescence will be produced to indicate safety. To avoid liquid bacteria culture handling and enhance the efficiency of the sensing process, we decide to immobilize biosensor cells in PEG-PVA hydrogel discs as prototypes.
With the hardware design of cell immobilization, we need to make sure the PEG-PVA polymer matrix would not negatively affect the fluorescence signal output of cells. We wonder whether colour signals could be blocked by the matrix or production rate affected because of the solid culture medium.
Key Achievements
- To determine the culture to gel volume ratio that has optimal fluorescence viability but not affecting the structure and strength of hydrogel matrix
- To investigate fluorescence behavior of the hydrogel biosensor disc
- To determine how usage of gel as a biosensor hardware could affect the viability of fluorescence signal
- To determine whether adding LB medium as nutrient supply could affect the viability of fluorescence signal
- Bacteria Culturing medium
- GFP expressing E. coli Cells in LB medium VS in PVA Gel
- RFP expressing E. coli Cells in LB medium VS Cells in PVA Gel
- Nutrient Supply VS Without Nutrient Supply: LB Medium
- Cell Culture to Gel Volume Ratio
- 200 μL:1300 μL
- 200 μL:1300 μL
- Blue Light Transilluminators Result on photo taken by phone
camera
- Red Fluorescent Protein viability on photos
- Green Fluorescent Protein viability on photos
- Temperature control: 37.0 °C
- Size of gel(12 well plates as mold): diameter=20 mm, height=3mm
- Nutrients supply (LB applied)
- No antibiotics added
- No pH tuning
Independent Variables (See Figure 12 for 12 well plate design):
Dependent Variables
Controlled Variables
In Figure 11, we can see that 100 μL E. coli GFP is too little to be evenly distributed in the PVA gel while 200 μL E. coli GFP is good enough to see the evenly distributed Fluorescence in 1.5 ml PVA 1799 Gel
Thereby,
From the picture above taken by on Blue Light Transilluminators, it could be conclude that:
- Using 200μL GFP Cell Culture to 1300μL Gel Volume as ratio
- There is distinguishable red colour of RFP gel evenly distributed in the disc
- There is distinguishable green colour of RFP gel evenly distributed in the disc
- There is a higher viability of fluorescence (GFP and RFP) in PVA hydrogel, compared to the in liquid culturing medium, meaning that gel is a better medium for viable result inspection.
- As adding LB nutrient to hydrogel does not have observable difference to that not added LB, we can add LB for better culturing conditions(eg. activation of hydrogel biosensor) , as well as assuming all the fluorescence comes from fluorescent protein instead of LB.
Experiment 3: Detection of Valinomycin (Analogue of Cereulide) using engineered B. subtilis cells Hydrogel
Overview
To prevent potential leakage and avoid liquid bacteria culture handling for users, in our hardware design, B. subtilis cell cultures are firstly sporulated, then immobilized in PEG-PVA hydrogel.
Cereulide is hydrophobic, meaning that it would be difficult for it to diffuse and reach cells in liquid medium and trigger circuits to give signals. Based on our research, PVA hydrogel-based cell biosensors had been applied by researchers to detect hydrophobic toxins[4]. Another advantage of using hydrogel mentioned in literature[5] is that, cell immobilization in hydrogel could improve the sensor-toxin assembly by avoiding diffusion of cell culture, facilitating close contact between the cells biosensor and analyte,resulting in improved detection limits
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Before biosensor engineering, we had concerns about whether performance of the biosensor cells would be affected by the gel matrix. After consulting with Professor Sun Fei of the HKUST Chemical and Biological Engineering(CBE) Department, who specializes in Bioengineering and Biomaterial development, the following advice solved our concerns:
- B. subtilis can survive under anaerobic environment so capsules in hydrogel solution, or even anaerobic capsule design would not affect their growth and function.
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Cells are robust enough to be immobilized in gel and be reactivated with nutrient supply, fluorescence protein signal would not be affected much as well
- Sporulation of B. subtilis for embedding might not be necessary.
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During the engineering process, we embedded Cycle 0 B.subtilis Cells engineered and tested working into gel. However, the fluorescence signals were not significantly given out upon adding of valinomycin. Therefore, we also consulted Mr. Kachin Wong, CEO of SPES Tech, who specialized in hydrogel cells culturing. We are grateful for the following list of advice from him:
- The scale of biosensor and the concentration of cells could be factors contributing to insufficient fluorescence
- For better signal from plate reader scanning:
- Try making gels with smaller diameter but keeping the same volume of gel-cell mixture. You can try using smaller mold, it could be 24 well plates, instead of 12 well plates.
- Try using cell culture with higher OD before adding to gel solution
We then adjusted the protocol by using cells that had been incubated for a longer time and made gels with in 24-well plate with the same volume of gel-cell mixture(1500uL), and we successfully obtained result that proves fluorescent signal could significantly increase upon 3uM Valinomycin added.
Key Achievements
- To determine the time required from adding valinomycin to a significant rise in fluorescent signal
- To determine stability of B. subtilis Cells Hydrogel and spores Hydrogel
- To determine whether spores can germinate in hydrogel
- 0μM vs 0.3 μM vs 3 μM Valinomycin
- Cells in LB medium VS Cells embedded in PVA Gel
- Spores in LB medium VS Cells embedded in PVA Gel
- Red Fluorescence Intensity [Ex/Em: 607 nm/584 nm (Cross Well Scan)]
- Time intervals (scan every 20 mins)
- Temperature: 37.0℃, No Shaking
- pH: 8.5
- Antibiotics: Chloramphenicol CHL(added according to our protocol)
- Cells Culture(same for spores): 200 µl 36 hrs Engineered cycle 0 cells
- Size of gel(24 well plates as mold):diameter=12mm, height=10mm
- Nutrients supply (LB applied)
Independent Variables(See Figure 15 for 24 well plate design)
Dependent Variables
Control Variables
We follow the Whole-Cell Hydrogel B. subtilis Biosensor Construction
Protocol
to make gels with the following settings:
Gel Making:
- Used 24 well-plates as molds
- Cells culture we used: Picked colony from agar plate and inoculate in 22 ml LB medium for 36 hours (50ml Falcon tubes for sufficient aeration)
- Spores culture we used: Engineered cycle 0 cells which were sporulated for 6 days (centrifuge the cells for 14000 rpm for 1.5 mins, then resuspended in water)
- Tune pH with 10M NaOH, test with pH paper until it reaches 8.5 for optimal
- B. subtilis Cell Gel and Spore Gels were premade and stored in 4°C fridge for 5 days. Reactivation is done by putting the gels into 37°C incubator for 1 hour before adding valinomycin.
The Four Columns correspond to cells/spores in either LB medium or PVA gel solution The 1st row correspond to LB or PVA gel without any cell culture as blank, The 2nd, 3rd, and 4th rows correspond to reaction between bacteria and 3 different concentration of valinomycin (0μM, 0.3 μM, 3 μM)
Calculation for valinomycin adding to obtain certain concentration:
Master mix is made for valinomycin for these 4 concentrations respectively
Figure 17 shows that 2 hours after adding 3μM Valinomycin to the biosensor cell gel, the fluorescent signal significantly increases from 270 units (at t= 1 hr 40 mins) to 657 units at (t=2 hr) and stay between the range of 600-666 units (from t=2 hr to 7 hr).
Figure 18 demonstrates that our Circuit Design team conducted a similar experiment using the same bacteria (cycle 0) in BHI Liquid Medium. The significant fold change in fluorescence intensity was observed 4 hours after adding 3μM Valinomycin. However, when using hydrogel, the time required for the signal to appear was reduced by 2 hours.
Figure 19 shows that, although spores embedded in gel do not show significant increase in fluorescent signal, from sampling and observation on microscope, it is found that spores can germinate in hydrogel after 31 hours incubation with nutrients (LB) added.
It could be concluded that biosensor cells' performance would not be negatively influenced, instead, biosensors embedded in gel could have a better performance in detecting valinomycin(analogue of cereulide) compared to liquid medium. This means that our engineering direction is correct that we had improved detection limits of hydrophobic substances by tuning the hydrophobicity of the cell test kit to allow better interaction between analyte and biosensor.
Although the red fluorescence signal could only be detected in plate reader, and not yet proved intense/strong enough to be viable in blue light transilluminator, we could still see the advantage of using gel as a biosensor hardware for higher viability of fluorescence, by comparing the signal output efficiency between cells in liquid culturing medium and hydrogel.
References
- [1] L. D. Knechtet al., “Bacterial spores as platforms for bioanalytical and biomedical applications,” Analytical and bioanalytical chemistry, vol. 400, no. 4, pp. 977-989, 2011, doi: 10.1007/s00216-011-4835-4.
- [2] Schulz-Schönhagen, K. (2019). Bacillus subtilis biosensors: Engineering a living material sensor platform (Doctoral dissertation, ETH Zurich).
- [3] L. Delbrassinne et al., “Determination of Bacillus cereus Emetic Toxin in Food Products by Means of LC-MS,” Food analytical methods, vol. 5, no. 5, pp. 969-979, 2012, doi: 10.1007/s12161-011-9340-z.
- [4] T. Fine et al., “Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection,” Biosensors & bioelectronics, vol. 21, no. 12, pp. 2263-2269, 2006, doi: 10.1016/j.bios.2005.11.004.
- [5] E. Wahid et al., “Biological and technical challenges for implementation of yeast-based biosensors,” Microbial biotechnology, vol. 16, no. 1, pp. 54-66, 2023, doi: 10.1111/1751-7915.14183.