Team:Bolivia/LaboratoryProtocols

TEAM BOLIVIA

LABORATORY PROTOCOLS

Here we explain in more detail the protocols we plan to carry out in the lab for the different the phases of the experimental design.

I. Competence induction and transformation:

1. Materials and reagents:

  • Sterile distilled water

  • Calcium chloride (anhydrous)

  • LB agar plates supplemented with appropriate antibiotic (Ampicillin: 100 μg/mL or Kanamycin: 50 μg/mL)

  • E. coli DH5α or BL21

  • Incubator with shaker

  • Centrifuge

  • Spectrophotometer

  • Water bath or heating block

  • Eppendorf tubes

  • PCR tubes (200 μL)

  • Metal bacteriological loop

  • Drigalski loop

2. Procedure
E.coli culture:

  • Take a colony and inoculate it in 5 mL of unsupplemented LB broth.

  • Incubate with shaking (160 RPM) 18 - 24h at 37°C.

Preparation of CaCl2 solutions (for 50 mL 1M "stock solution"):

  • Weigh 5.05g of anhydrous calcium chloride.

  • Add 50 mL of distilled water

  • Mix until all the calcium chloride is dissolved.

  • Sterilize in an autoclave

Preparation of CaCl2 solutions (for 50 mL 0.1M )

  • In a 50 mL falcon tube add 5 mL of the 1M solution and add 45 mL of sterile distilled water.

Generation of competent cells:

  • Take 1 mL of the previous culture and inoculate it in another tube with 5 mL fresh LB broth and incubate for 3h/37°C with shaking or until it reaches OD600 0.4 - 0.6.

  • Pipette 1.5 mL of the culture into an Eppendorf tube and place on ice for 20 min.

  • Centrifuge at 4000 RPM/10 min

  • Discard supernatant and resuspend with 1 mL of ice-cold 0.1M CaCl2 solution.

  • Place on ice for 30 min.

  • Centrifuge at 4000 RPM/10 min.

  • Discard supernatant and resuspend with 200 μL of ice-cold 0.1M CaCl2 solution.

  • Aliquot 50 μL into 200 μL PCR tubes.

  • Add 1 μL of plasmid (1 μg/μL), if the concentration is lower, a higher volume must be added.

Heat-shock transformation:

  • Aliquot 50 μL of the above solution into 200 μL PCR tubes.

  • Add 1 μL of plasmid (1 μg/μL), if the plasmid concentration is lower, a higher volume has to be added.

  • Place on ice for 30 min.

  • Place at 42°C for 30 seconds.

  • Place on ice for 2 min.

  • Add 250 μL of unsupplemented LB broth.

  • Incubate with shaking (225 RPM) for 1h/37°C.

  • Plate on solid LB agar medium supplemented with the appropriate antibiotic (200 μL/box and 20 μL/box).

  • Incubate plates at 37°C/24h and observe results.

II. Biosensor arsenic induction (semi-quantitative):

1. Materials and reagents:

  • Materials and reagents

  • LB broth (0.2% glycerol)

  • Boxes with LB agar supplemented with Kanamycin (50 μg/mL)

  • Sodium arsenite stock solutions (40X)

  • (20 - 200 - 400 - 600 - 800 - 2000 - 4000 ppb)

  • Kanamycin stock solution (5 mg/mL)

  • Distilled water

  • Falcon tubes (25 mL - 50 mL)

  • Eppendorf tubes

  • Incubator with shaker

2. Procedure:

  • Grow the biosensor to be characterised on fresh LB agar supplemented with kanamycin (50 μg/mL).

  • Take an isolated colony and inoculate it in 25 mL of liquid medium supplemented with kanamycin (200 RPM).

  • Incubate 24h/37°C with constant shaking (200 RPM).

  • Measure OD600 of the culture.

  • Dilute in supplemented liquid medium to OD600 = 0.025.

  • Dispense 1 mL into each eppendorf tube.

  • Load 25 μL of the inducer prepared the previous day into each corresponding tube (1/40 dilution).

  • Incubate 24h/37°C with shaking (200 RPM).

  • Observe colour generation and analyse results.

III. Biosensor lyophilization on paper strips:

1. Materials and reagents:

  • LB broth (0.2% glycerol)

  • Boxes with LB agar supplemented with Kanamycin (50 μg/mL)

  • Buffer solution (0.5% peptone, 0.3% meat extract, 10% gelatine, 1% sodium ascorbate, 5% raffinose, and 5% sodium glutamate)

  • Filter paper or M3 paper

  • Polypropylene tubes (falcon)

  • Freeze dryer

  • Centrifuge

  • Incubator with Shaker

  • Sodium arsenite stock solutions (1X)

  • (0.5 - 5 - 10 - 15 - 15 - 20 - 50 - 100 ppb)

2. Procedure:

  • Grow the biosensor to be lyophilised on fresh LB agar supplemented with kanamycin (50 μg/mL).

  • Take an isolated colony and inoculate it in 5 mL of liquid medium supplemented with kanamycin (200 RPM).

  • Centrifuge cultures 3500g/6 min and discard supernatant.

  • Resuspend in 1 mL of pre-warmed (37°C) protective solution.

  • On 0.5 x 5 cm paper strips deposit 5 μL spots.

  • Dry in laminar flow hood for 5 min in a polypropylene tube.

  • Dry in a freeze dryer according to the following programme: 4 mbar/2h, 0.4 mbar/2h, 0.04 mbar/2h.

  • For analyte exposure tests dip the strip in 5 mL of arsenic solution for 30 min/36°C.

  • Remove and incubate until colour development.

IV. Biosensor arsenic induction (quantitative):

1. Materials and reagents:

  • LB broth (0.2% glycerol)

  • Boxes with LB agar supplemented with Kanamycin (50 μg/mL)

  • Metal stock solutions (40X)

  • Arsenic (10 μM)

  • Magnesium (4000 μM)

  • Iron (4000 μM)

  • Mercury (80 μM)

  • Kanamycin stock solution (5 mg/mL)

  • Distilled water

  • Falcon tubes (25 mL - 50 mL)

  • Eppendorf tubes

  • Incubator with shaker

  • Spectrophotometer

2. Procedure:

  • Grow the biosensor to be characterised on fresh LB agar supplemented with kanamycin (50 μg/mL).

  • Take an isolated colony and inoculate it in 25 mL of liquid medium supplemented with kanamycin (200 RPM).

  • Incubate 24h/37°C with constant shaking (200 RPM).

  • Measure OD600 of the culture.

  • Dilute in supplemented liquid medium to OD600 = 0.025.

  • Dispense 2 mL into each eppendorf tube.

  • Load 50 μL of the inducer prepared the previous day into each corresponding tube (1/40 dilution).

  • Incubate 24h/37°C with shaking (200 RPM).

  • Take absorbance readings at 400 nm in a spectrophotometer.

  • Analyse results.

V. Biosensor induction with other metals (specificity):

1. Materials and reagents:

  • LB broth (0.2% glycerol)

  • Boxes with LB agar supplemented with Kanamycin (50 μg/mL)

  • Metal stock solutions (40X)

  • Arsenic (10 μM)

  • Magnesium (4000 μM)

  • Iron (4000 μM)

  • Mercury (80 μM)

  • Kanamycin stock solution (5 mg/mL)

  • Distilled water

  • Falcon tubes (25 mL - 50 mL)

  • Eppendorf tubes

  • Incubator with shaker

  • Spectrophotometer

2. Procedure:

  • Grow the biosensor to be characterised on fresh LB agar supplemented with kanamycin (50 μg/mL).

  • Take an isolated colony and inoculate it in 25 mL of liquid medium supplemented with kanamycin (200 RPM).

  • Incubate 24h/37°C with constant shaking (200 RPM).

  • Measure OD600 of the culture.

  • Dilute in supplemented liquid medium to OD600 = 0.025.

  • Dispense 2 mL into each eppendorf tube.

  • Load 50 μL of the inducer prepared the previous day into each corresponding tube (1/40 dilution).

  • Incubate 24h/37°C with shaking (200 RPM).

  • Take absorbance readings at 400 nm in a spectrophotometer.

  • Analyse results.

VI. Biosensor induction with real samples:

1. Materials and reagents:

  • LB broth (0.2% glycerol)

  • Boxes with LB agar supplemented with Kanamycin (50 μg/mL)

  • Well water samples (OTBs)

  • Kanamycin stock solution (5 mg/mL)

  • Distilled water

  • Falcon tubes (25 mL - 50 mL)

  • Eppendorf tubes

  • Syringe filter (0.22 μm)

  • Incubator with shaker

  • Spectrophotometer

2. Procedure:

  • Sterilise 10 mL of sample by syringe filter filtration.

  • Culture the biosensor to be characterised on fresh LB agar supplemented with kanamycin (50 μg/mL).

  • Take an isolated colony and inoculate it in 20 mL of liquid medium supplemented with kanamycin (200 RPM).

  • Incubate 24h/37°C with constant shaking (200 RPM).

  • Measure OD600 of the culture.

  • Dilute in supplemented liquid medium to OD600 = 0.025.

  • Dispense 2 mL into each eppendorf tube.

  • Load 500 μL of the sterile sample into the corresponding tubes (1/4 dilution).

  • Incubate 24h/37°C with shaking (200 RPM).

  • In a spectrophotometer take absorbance readings at 400 nm and extrapolate to calibration curve to quantify arsenic concentration.

  • Analyse results.

  • For semi-quantitative determination dip the strip with the freeze- dried biosensors in 5 mL of sterilised sample for 30 min/36°C.

  • Remove and incubate at 37°C until colour development.

Water sampling manual

Our team designed a biosensor to detect arsenic in drinking water, which in many cases is found in wells that supply water to different populations in Cochabamba. We developed a well-water sampling manual where considering the experimental design and the external services involved we explain the steps to be followed. This manual will be used by our team for the development of phase 5 of the experimental design “Biosensor response with real samples”.

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REFERENCES

[1] Bernard, E., & Wang, B. (2017). Synthetic Cell-Based Sensors with Programmed Selectivity and Sensitivity. Biosensors And Biodetection, 349-363.

https://doi.org/10.1007/978-1-4939-6911-1_23

[1] Stocker, J., Balluch, D., Gsell, M., Harms, H., Feliciano, J., & Daunert, S. et al. (2003). Development of a Set of Simple Bacterial Biosensors for Quantitative and Rapid Measurements of Arsenite and Arsenate in Potable Water. Environmental Science & Technology, 37(20), 4743-4750.

https://doi.org/10.1021/es034258b

[3] Wan, X., Volpetti, F., Petrova, E., French, C., Maerkl, S., & Wang, B. (2019). Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals. Nature Chemical Biology, 15(5), 540-548.

https://doi.org/10.1038/s41589-019-0244-3