Protocol for competent cells

Objective:

To generate competent E. coli DH5α cells, enabling them to uptake genetic material through chemical methods (heat shock method).

Materials:

Biological Materials:

Reagents:

Labware:

Equipment:

Procedure:

Day 1 (afternoon/evening): Prepare an overnight culture
  1. Place the sterile LB broth (10 ml in a tube) and the E. coli DH5α bacteria (either in the Eppendorf tube or on the LB agar plate) in a rack.
  2. Add 0.1 ml (100 µL) of E. coli DH5α to the 10 ml of sterile LB broth.
  3. Incubate the tube overnight (approximately 12 hours) at 37°C with shaking at 200 rpm.
Day 2 (morning): Ensure you have crushed ice for the culture and CaCl2.
  1. Remove the overnight culture and measure the OD600 (should be >1). To do this, add 10 µL of the E. coli DH5α culture to a spectrophotometer cuvette and 900 µL of LB broth. Record the reading and correct for dilution.
  2. Calculate the necessary dilution to reach an OD600=0.1. Transfer the calculated volume to the tube with 10 ml of LB broth using a micropipette.
  3. Incubate the tube at 37°C with shaking at 200 rpm until an OD600=0.5-0.7 is reached (approximately 2 hours).
  4. Transfer 1 ml of the culture to an Eppendorf tube and place on crushed ice (4°C) for 20 minutes.
  5. Centrifuge the Eppendorf tubes for 1 minute at 13,000 rpm. Remove the tube, discard the supernatant, and place it back on crushed ice.
  6. Slowly add 500 µL of cold CaCl2 (150 mM) to the tubes. Gently invert the tubes 4 times to resuspend the pellet.
  7. Incubate on ice for 20 minutes.
  8. Centrifuge the Eppendorf tubes for 1 minute at 13,000 rpm. Remove the tube, discard the supernatant, and place it back on crushed ice.
  9. Slowly add 50 µL of cold CaCl2 (150 mM) to the tubes. Gently invert the tubes 4 times to resuspend the pellet.
  10. Store at 4°C for immediate use or at -20°C for use within the week.

Notes:

The cells are now ready for use in the transformation protocol. If long-term storage at -80°C is required, prepare CaCl2 (150 mM) with 20% glycerol and use it in step #12.

Reference

Green, M. Richard, & Sambrook, J. Frank. (2012). Molecular cloning : A laboratory manual. 4th ed. Cold Spring Harbor (N.Y.): Cold Spring Harbor Laboratory Press.

Competent Cells Protocol

1

Day 1 (afternoon/evening): Prepare an overnight culture
1. Add 0.1 ml E. coli DH5a to 10 ml LB broth.
2. Incubate overnight at 37°C, 200 rpm.

2

Day 2 (morning): Measure OD600 of culture (>1).
Dilute to OD600-0.1 in 10 ml LB.

3

Incubate at 37°C with
shaking until
OD600=0.5-0.7 (~2 hrs).

4

Transfer 1 ml of culture to an Eppendorf tube and place on crushed ice (4°C) for 20 minutes. Centrifuge at 13,000 rpm for 1 min, discard supernatant.

5

Add 500 μL cold CaCl2 (150 mM), invert to resuspend. Incubate on ice for 20 min, centrifuge again, discard the supernatant.
20 min

6

Add 50 μL cold CaCl2 (150 mM), resuspend. Store at 4°C (short-term) or -20°C (1 week).
CaCl2 (150 mM)

Bacterial Transformation Protocol

Objective:

To transform DH5-alpha Thermo ScientificⓇ cells with mercury-detecting plasmids.

Prerequisites

Procedure

  1. Autoclave Materials: Prepare LB liquid medium.
  2. FlasAfter autoclaving, gently swirl the medium to mix the agar evenly. Cool the medium to around 50°C, then add kanamycin/ampicillin at a concentration of 0.1 mg/ml. Mix well without creating bubbles, and pour approximately 15 ml of the medium into each plate.ks
  3. Once the medium has fully solidified, the plates are ready for use. Alternatively, invert the plates and store them at 4°C. To prevent contamination, perform the entire process using a Bunsen burner or within a laminar flow hood.
  4. For bacterial cell transformation with a plasmid, use competent E. coli DH5 cells, previously prepared with CaCl₂ and stored at -70°C in 100 μl aliquots.
  5. Thaw the cells on ice and add 50 μl to a ster
  6. ile Eppendorf tube.Add 1 μl of plasmid DNA and incubate on ice for 25-30 minutes.
  7. Subject the mixture to heat shock at 42°C for 90 seconds, then immediately place on ice for 2 minutes.
  8. Add 400 μl of LB broth without antibiotic and incubate at 37°C for 15 minutes in a water bath or incubator.
  9. Plate the cells on LB agar plates containing ampicillin (selection antibiotic) and spread them evenly with a Drigalski spatula. Perform plating with and without DNA on plates with and without antibiotic (4 plates in total).
  10. Incubate overnight at 37°C.
  11. The next day, check for the growth of ampicillin-resistant bacterial colonies.
  12. Calculate transformation efficiency, expressed as the number of transformants per microgram of DNA.

References

Green, M. Richard, & Sambrook, J. Frank. (2012). Molecular cloning : A laboratory manual. 4th ed. Cold Spring Harbor (N.Y.): Cold Spring Harbor Laboratory Press.

Transformation Protocol

1

Prepare LB agar plates: 

  1. Autoclave materials.
  2. Cool LB medium to 50°C, add 0.1 mg/ml antibiotic (Kanamycin/Ampicillin), and pour ~15 ml into each plate.
  3. Let plates solidify and store at 4°C (if not using mmediately).

2

Thaw competent cells:

  1. Thaw 50 μl of DH5-alpha cells on ice
  2.  Add 1 μl plasmid DNA to the cells and incubate on ice for 25-30 minutes.

3

Heat shock:
Heat shock at 42°C for 90 seconds, then place on ice for 2 minutes.

4

Recovery: Add 400 μl LB broth (no antibiotic) and incubate at 37°C for 15 minutes.

5

Plate cells:

  • Plate cells on LB agar with antibiotic, using a Drigalski spatula to spread evenly.
  • Prepare 4 plates: with and without DNA, with and without antibiotic.

6

Incubate: Incubate plates overnight at 37°C.
Results:

  • Check for growth of antibiotic-resistant colonies the next day.
  • Calculate transformation efficiency (transformants/μg DNA)

Protocol for Handling Mercury Compounds

Objective:

Ensure the proper handling of mercury compounds used during the validation of the mercury biosensor, including their storage, minimizing handling, contamination, and disposal of residuals.     

Prerequisites

Materials:

Reagents:

Mercury (II) chloride (CAS 7487-94-7) 
Density 5,44 g /cm³ at 20 °C
Color white
Melting point/freezing point 277 °C
Water solubility 74 g /l at 20 °C
Danger label(s)
Methylmercury(II) chloride (CAS 115-09-3)
Physical State Liquid
Danger label(s)

Equipment:

Personal Protective Equipment

Storage of Reagents

Storage of Reagents

Handling of Mercury Compounds

a) Weighing

b) Solution Preparation

c) Cleaning the Work Area

d) Spill Procedure

Procedure in Case of Contact with Compounds

The first step in the event of a laboratory accident is to implement established biosafety and containment protocols for mercury, and inform the responsible supervisor of the incident.

a) Mercury Salts Spilled on Skin

b) Inhalation of Mercury Salts

References

Leermakers, M., Lansens, P., & Baeyens, W. (1990). Storage and stability of inorganic and methylmercury solutions. Fresenius' Journal of Analytical Chemistry, 336, 655-662. https://doi.org/10.1007/BF00331410.
Ramírez, A. V. (2008, March). Intoxicación ocupacional por mercurio. In Anales de la Facultad de Medicina (Vol. 69, No. 1, pp. 46-51). UNMSM. Facultad de Medicina.
Basu, N., Bastiansz, A., Dórea, J. G., Fujimura, M., Horvat, M., Shroff, E., Weihe, P., & Zastenskaya, I. (2023). Our evolved understanding of the human health risks of mercury. Ambio, 52(5), 877–896. https://doi.org/10.1007/s13280-023-01831-6

Mercury Compounds Handling Protocol

1

Prerequisites

  • Obtain Safety Data Sheets (SDS) for mercury compounds.
  • Wear Personal Protective Equipment (PPE): gloves, goggles, lab coat, N95 mask.
  • Complete First Aid Training.

2

Work Area Preparation

  • Ensure fume hood is functioning.
  • Prepare neutralizing solution (Sodium Sulfide Na2S 0.5M).
  • Maintain controlled lab temperature to prevent volatilization

3

 Handling & Weighing Mercury Compounds

  • Weigh compounds in a fume hood, using glass or Teflon containers.
  • Clean balance with sodium thiosulfate solution.
  • Recalibrate balance after cleaning.

4

Solution Preparation

  • Use glassware exclusively for mercury solutions
  • Store methylmercury in Teflon containers for up to one month

5

Spill Procedure

  • Neutralize with sodium thiosulfate.
  • Prepare 4 plates: with and without DNA, with and without antibiotic.
  • Use mercury sponges to clean up.
  • Label and dispose of waste.

6

Emergency Protocol

  • Skin Contact: Wash for 15 minutes; use safety showers for large areas.
  • Inhalation: Move to fresh
  • air and seek medical

Encapsulation

Objective:

To encapsulate recombinant bacteria that detect inorganic and organic mercury in alginate gels to enhance their functionality and viability.

Reagents:

Materials:

Equipment:

Personal Protective Equipment:

Alginate Encapsulation Protocol

  • Weigh the high and low-viscosity sodium alginate to prepare a 2% (w/v) concentration in demineralized water.
  • Sterilize the solution at 117°C for 15 minutes.
  • Add the necessary amount of alginate to a bacterial culture with an approximate concentration of 10⁹ CFU/mL to achieve a final concentration of 10% (v/v).
  • Discharge the mixture of sodium alginate and bacterial culture through a peristaltic pump with a 0.5 mm diameter tubing at a flow rate of 3 mL/min into a 0.2 M CaCl₂ solution with continuous stirring (100 rpm).
  • Allow the mixture to stand in the 0.2 M CaCl₂ solution for 20 minutes or until firm structures are formed.
  • Wash the beads and suspend them in saline solution (0.9% w/v) until use.

References

Hernández-Gallegos, M., Javier Solorza-Feria, M., Velázquez-Martínez, J., Rodríguez-Huezo, M.,  Gutiérrez-López, M. & Hernández-Sánchez, H.( 2023) "Protective Effect of Alginate Microcapsules with Different Rheological Behavior on Lactiplantibacillus plantarum 299v" Gels 9, no. 9: 682. https://doi.org/10.3390/gels9090682

Mercury Compounds Handling Protocol