Mercury(II) Chloride Plate test protocol

7-30-14 Test

7-27-14 1. E. coli strains were streaked for singles on LB+Km plates for single colonies from -80 stocks. Strains tested: E. coli pBBRBB::mer, E. coli pBBRBB::gfp (vector control), E. coli pDU1358 (positive control).

7-28-14 2. Single colonies were grown in 5 mL LB+Km (50ug/mL) overnight

7-29-14 3. Overnight cultures were diluted 1/10 in minimal medium 4. 3 mL of diluted culture were spread evenly on Tryptone Medium plates. Three biological replicates were prepared for each strain.

Tryptone Medium per liter 15 g tryptone 5 g NaCl 10 g Noble Agar 1 pellet sodium hydroxide 1 L ddH2O

5. Plates were allowed to sit for 5 minutes after which excess culture was removed from each plate with a pipet

6. Plates were then allowed to dry for 10 minutes and filter disc was placed in the middle of each plate.

7. 10 uL of the stock 0.1M HgCl2 solution was then added to the filter disc. As a control, one plate was also prepared for each strain, and water was added to the filter to ensure nothing toxic was leaching from the filter disc.

8. Plates were incubated overnight at 37C

7-30-14

9. Zones of inhibition were measured as the diameter of clearing around the filter disc. Two measurements were made for each plate, and three plates were tested for each strain.

10-8-14 Test

10-5-14 1. Strains were streaked for singles on LB+Km plates for single colonies from -80 stocks. Strains tested: E. coli pBBRBB::mer, E. coli pBBRBB::gfp (vector control), E. coli pBBRBB::merΔmerA (to test for merPT transport and extra sensitivity of merA deletion strains to HgII), P. putida pBBRBB::mer, P. putida pBBRBB::gfp (vector control)

10-6-14 2. Single colonies were grown in 5 mL LB+Km (50ug/mL) overnight

10-7-14 3. Overnight cultures were diluted 1/10 in minimal medium 4. 3 mL of diluted culture were spread evenly on Tryptone Medium plates. Three biological replicates were prepared for each strain.

Tryptone Medium per liter 15 g tryptone 5 g NaCl 10 g Noble Agar 1 pellet sodium hydroxide 1 L ddH2O

5. Plates were allowed to sit for 5 minutes after which excess culture was removed from each plate with a pipet

6. Plates were then allowed to dry for 10 minutes and filter disc was placed in the middle of each plate.

7. 10 uL of the stock 0.1M HgCl2 solution was then added to the filter disc. As a control, one plate was also prepared for each strain, and water was added to the filter to ensure nothing toxic was leaching from the filter disc.

8. Plates were incubated overnight at 37C (E. coli) and 30C (P. putida).

10-8-14

9. Zones of inhibition were measured as the diameter of clearing around the filter disc. Two measurements were made for each plate, and three plates were tested for each strain.