These are methods to screen for and assay the activity of the common reporter enzyme β-Glucuronidase (GUS) activity. Because the catalytic activity of β-Glucuronidase is very similar to β-Galactosidase (LacZ) these protocols are also very similar to the LacZ protocols.
- Media of choice
- X-gluc stock solution (50mg/mL in DMF)
- Prepare your liquid media and add the desired amount of agar (usually 1-2%).
- Autoclave the media for the requisite time.
- Add 1.2μL of X-gluc stock solution for every mL of media (e.g. if making 1L of media add 1.2ml of stock solution)
- If desired, add antibiotic.
- Pour plates.
- Cultured Cells
- Suspension Buffer (50mM NaH2PO4
- X-gluc stock solution (50mg/mL)
- Premeabilization Solution (9:1 acetone to toluene (v/v))
- Pellet 1ml of culture by centrifugation
- Discard the supernatant and resuspend the pellet in 400μL Suspension Buffer
- Add 25ul of Permeabilization Solution to cell suspension.
- Incubate at 37°C for 30-60 minutes.
- Add 5μL of X-gluc stock solution.
- A green/blue color should develop shortly in positive cultures.
- Depending on what cells you're using, the solvent mix may not permeabilize your cells. In such a case you might attempt using 25μL of chloroform or 12.5μL of 1%SDS or both.
- Suspension Solution (50mM NaH2PO4)
- Permeabilization solution (9:1 acetone to toluene (v/v))
- GUS Buffer (50mM NaH2PO4, 10mM β-mercaptoethanol, 1mM EDTA and 0.1% Triton X-100)
- 4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM NaH2PO4)
- Stop Buffer (200mM Na2CO3)
- Measure and record the OD600 of the cell culture
- Pellet 1mL of culture by centrifugation.
- Resuspend the pellet in 400μL of Suspension Solution.
- Add 25ul of permeabilization solution.
- Incubate for 30 to 60 minutes at 37°C
- Take 50μL of this cell suspension and add it to 200μL of GUS Buffer.
- Add 20μL of 4-NPG stock solution.
- Let the reaction run for 10-30 minutes.
- Add 200μL Stop Solution to halt the reaction.
- Centrifuge to pellet cell debris.
- Measure the OD405 of the stopped reaction.
- Use this one for E. coli.
- 100mM sodium phosphate buffer (pH=7)
- 40mM NaH2PO4
- 60mM Na2HPO4
- 0.1M potassium chloride solution
- 10mM magnesium sulfate solution
- 1M Na2CO3
- 4-Nitrophenyl β-D-glucuronide (4-NPG) stock solution (10mg/ml in 50mM sodium phosphate buffer (pH=7)) only make 1mL of this!!!
- 10% Triton X-100 (in water)
- 1. Grow culture until OD600 is between 0.6 and 1.0
- 2. Prepare 10mL of GUS Buffer (measures 10 samples) by adding:
- 5mL of sodium phosphate buffer (pH=7)
- 3mL H2O
- 1mL of potassium chloride solution
- 1mL of magnesium sulfate solution
- 35μL β-mercaptoethanol
- 20mg Lysozyme
- 3. Pellet 1.5 ml of culture by centrifugation for 1 minute.
- 4. Resuspend in 1ml 100mM sodium phosphate buffer.
- 5. Pellet again by centrifugation.
- 6. Resuspend in 750μL GUS buffer.
- 7. Vortex briefly to mix.
- 8. Incubate for 30 min in 37°C water bath.
- 9. Add 8ul of 10% Triton-X.
- 10. Vortex briefly and incubate on ice for 5 mins.
- 11. Add 80ul of 4-NPG solution and start the timer.
- 12. Incubate in 37°C water bath.
- 13. When the color is clearly yellow (between 10 and 30 mins), stop reaction by adding 300μL 1M Na2CO3
- 14. Record the time.
- 15. Centrifuge the reaction for 1 minute at full speed.
- 16. Measure the OD405 of the supernatant.
Use this one for Lactobacillus spp.