$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Exposure Assay
- Designing a plate layout.
NOTE: Be sure to have all pipetting values calculated and stocks prepared prior to starting the exposure assay. How to properly design an experiment and what controls to include is detailed in the text. In addition, the experiment should not be started if there is oxygen in the anaerobic chamber indicated on the anaerobic monitor. - Design the plate layout according to a 96-well template. This will allow for 32 different treatments to run experiments in technical replicates of 3, which is best represented by a 4 x 8 grid to set up vials (see Figure 1).
NOTE: When testing for the role of a variable on Hg uptake with the mercury inducible biosensor, two treatments are required for each variable: the treatment (biosensor + mercury (Hg) + variable + nitrate) and its treatment blank (biosensor + variable + nitrate). When testing for the role of a variable on the physiology of the cell using the constitutive biosensor, two treatments are required for each variable: the treatment (biosensor + Hg + variable + nitrate) and the treatment blank (biosensor + variable + Hg). Mercury may be replaced with cadmium (Cd). Hg or Cd will become the variable when performing a calibration curve. The constitutive and inducible biosensors do not need to be run at the same time (in the same plate layout). A template example for the plate layout and corresponding 4 x 8 grid when testing a concentration range of magnesium (variable) is provided in Table 1.
2. Set up the 4 x 8 grid according to the assay plate layout.
1. Place 7 mL polytetrafluoroethylene (PTFE) standard vials in the tray.
NOTE: PTFE vials should be acid-washed or heat-sterilized prior to use. They should only be handled by manipulating the outside of the vial.
2. To each vial, add the exposure medium volume corresponding to each treatment.
NOTE: In an exposure with a total volume of 2,000 µL (2 mL), the added exposure medium will be: exposure medium µL = 2,000 µL – treatment (blank) µL. (e.g., exposure medium µL = 2,000 – 100 µL (biosensor) – 40 µL (nitrate) – 100 µL (Hg) – 100 µL (variable (e.g., magnesium sulfate, MgSO4)) = 1660 µL). Be sure that the volume added of the tested variable does not exceed 5% (100 µL) of the final volume.
3. Add the corresponding volume of the solution of the chemical variable to be tested to each vial, according to the plate layout.
4. Add nitrate to each vial so that the final concentration is 200 µM (40-80 µL). Exclude this step for constitutive biosensor treatment blanks.
5. Add Hg (5 nM when testing for a variable) or Cd (300 nM when testing for a variable) to the vials according to the plate layout. Exclude this step for mercury-inducible biosensor treatment blanks.
1. When using Hg, take the 4-8 µM stock and shake well. Dilute the solution in exposure medium in a 7 mL PTFE vial to 100-250 nM to make a working Hg solution. From this working solution, add Hg to the required vials. In this case, a calibration curve of Hg ranging from 0 to 12.5 nM.
NOTE: When testing for a variable’s effect on Hg or Cd bioavailability, make sure that the [Hg] or [Cd] remains constant across all treatments. When adding Hg or Cd, be sure to use one pipette tip, but never touch the exposure medium in the vials.
Shake in an orbital motion manually.
NOTE: The experiment may be paused now depending on the time required for Hg or Cd to speciate in solution. If left for more than an hour, place PTFE caps on the PTFE vials to prevent evaporation/contamination.
Gently pipette Biosensor Stock back and forth to ensure homogeneity. Add 100 µL of Biosensor Stock to each vial. Shake orbitally manually.
Prepare the plate reader to warm up to read with the following criteria: Temperature at 37˚C, kinetic run for 10 hours with reads every 2.5-5 minutes with orbital shaking in between reads, and fluorescence measurements with a fluorescence excitation of 440 nm and an emission of 500 nm.
Pipette 200 µL from each PTFE vial in the 4 x 8 grid into the corresponding wells of the 96-well plate (Black, 96-Well Clear-Bottom Nonbinding Surface Microplates). Pipette back and forth five times before transferring each 200 µL.NOTE: Instead of discarding the pipette tip, leave the pipette tip in the PTFE vial to keep track of pipetting progress.
Place the 96-well plate into the tray of the plate reader, then place the lid on the 96-well plate and begin the assay.
Table 1: An example plate layout for using the biosensor to test Hg bioavailability (5 nM) over a gradient of Magnesium (0-10 mM)
| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| a | Hg induced biosensor + Hg + Nitrate + 0 mM Mg | Hg induced biosensor + Nitrate + 0 mM Mg | Constitutive biosensor + Hg + Nitrate + 0 mM Mg | Constitutive biosensor + Hg + 0 mM Mg |
| b | Hg induced biosensor+ Hg + Nitrate + 0.1 mM Mg | Hg induced biosensor + Nitrate + 0.1 mM Mg | Constitutive biosensor+ Hg + Nitrate + 0.1 mM Mg | Constitutive biosensor + Hg + 0.1 mM Mg |
| c | Hg induced biosensor + Hg + Nitrate + 1 mM Mg | Hg induced biosensor + Nitrate + 1 mM Mg | Constitutive biosensor + Hg + Nitrate + 1 mM Mg | Constitutive biosensor + Hg + 1 mM Mg |
| d | Hg induced biosensor + Hg + Nitrate + 10 mM Mg | Hg induced biosensor + Nitrate + 10 mM Mg | Constitutive biosensor + Hg + Nitrate + 10 mM Mg | Constitutive biosensor + Hg + 10 mM Mg |