Quantification of Mercury bioaccumulated by metal binding protein (MBP) in recombinant DH5α induced by different Hg concentrations.

Reagents:

- Mercury chloride 20mg/ml;

- Chloramphenicol 34 µg/ml;

- Luria-Bertani + Mercury (LM) medium;

- Luria-Bertani (LB) medium;

- TN Buffer: Nacl 0.15M + Tris HCl 10mM.

Steps:

1. Inoculate bacterium (DH5α transformed with BBa_K1355003; DH5α transformed with BBa_K1355002 [control]) in LB liquid + chloramphenicol 34 µg/ml.

2. Incubate in shaker overnight at 37 ° C.

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

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

5. Aliquot 400 µl of the bacterial suspension in 5 tubes (1.5ml)

6. Add the amount of mercuric chloride necessary in the tubes to achieve the concentrations: 0,5 µg/ml, 0,25 µg/ml, 0,1 µg/ml, 0,05 µg/ml; and destine a tube for the zero Hg concentration.

7. 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. Recover the supernatant LM medium for quantification of Hg (Supernatant 1).

- Add 400 µl of TN Buffer to the pellet, re-suspend.

- Centrifugation at 12000g for 3 minutes. Recover the supernatant of TN for the measurement of Hg (Supernatant 2).

- Add 400µl of TN buffer to the pellet. Re-suspend Bacteria.

From here the samples are ready for analysis!

8. Add 50 ul of the bacterium re-suspended in TN in a white plate to measure the optical density in the spectrophotometer.

9. Destine 350 µl of each micro tube (the supernatant 1, supernatant 2 and bacterium re-suspended in TN) to quantify Hg in the Direct Mercury Analyzer (DMA-80).