Quantification of Green Fluorescent Protein (GFP) induced by different concentrations of mercury in Escherichia coli DH5α

Reagents:

- Chloramphenicol 34µg/ml;

- Mercury chloride 20mg/ml;

- Luria-Bertani + Mercury (LM) medium.

- TN Buffer (0.15M NaCl + 10mM Tris-HCl)

Steps:

1. Pre-inoculum: Inoculate bacteria (DH5α transformed with BBa_K1355002) in Luria-Bertani (LB) liquid + chloramphenicol 34 µg/ml. As positive control, inoculate DH5α transformed with p26G plasmid in LB liquid + ampicillin 100 µg/ml.

2. Incubate in the shaker at 37 °C overnight.

3. Transfer 1ml of bacterial suspension to 50 ml of LM without antibiotic.

4. Incubate in 37 °C shaker until the absorbance at 600nm spectrophotometer 0.4 to 0.6.

5. Before starting the induction with mercuric chloride, measuring fluorescence on spectrofluorometer (excit. 350nm, emission 500nm wavelength)

6. Aliquot 500ul of bacterial suspension in six tubes (2ml).

7. Add the amount of mercuric chloride to achieve concentrations: 1 ug/ml; 0,2 ug/ml; 0,1 ug/ml; 0,02ug/ml; 0,01ug/ml and a tube without mercury as a negative control.

8. Incubate at 37 °C in a shaker. After the scheduled time of growth in the shaker, the samples go through the following procedures:

- Centrifugation at 12000g for 3 minutes. Discard the supernatant

- Add 1 ml of TN buffer (0.15M NaCl + 10mM Tris-HCl) and re-suspend pellet.

- Centrifugation at 12000g for 3 minutes. Discard the supernatant.

- Add 500 ul TN buffer in the pellet.

From here the samples are ready to be analyzed by spectrofluorometer.

9. Add 100ul of each tube in black plate to measure in the spectrofluorimeter the intensity of GFP and white board to measure optical density on the spectrophotometric simultaneously. Making procedure doubled. Do not forget to add the positive control and negative control (DH5α with BBa_K1355002 without induction of mercury).

10. Measure O.D and GFP intensity at intervals of 60 minutes until 4.5 hours. Between intervals, incubated at 37 °C in the shaker.