This study presents a method for detecting bioavailable mercury using genetically engineered biosensors in an anaerobic environment. The approach utilizes magnesium sulfate to form non-bioavailable mercury complexes, allowing for the quantification of free mercury ions through fluorescence measurements.
Begin with an assay setup containing vials placed inside an anaerobic chamber.
Each vial contains media supplemented with sodium nitrate, mercury ions, and varying concentrations of magnesium sulfate.
Rotate the setup to mix the components, then incubate.
During incubation, magnesium sulfate promotes the formation of non-bioavailable mercury complexes, reducing free, bioavailable mercury ions in a concentration-dependent manner.
Next, add induced biosensors, genetically engineered bacteria carrying a plasmid, and mix.
The plasmid contains a mercury-responsive transcriptional regulator and a promoter linked to a fluorescent reporter gene.
Under anaerobic conditions, the bacteria utilize nitrate for respiration.
Free mercury ions enter the cells, bind the transcriptional regulator, and activate the fluorescent gene, producing fluorescent proteins.
Transfer aliquots from each vial into a multi-well plate.
Using a plate reader, measure the fluorescence intensity.
A decrease in fluorescence with increasing magnesium sulfate concentrations confirms the biosensor’s ability to detect changes in bioavailable mercury.
Before starting the experiment check the anaerobic monitor to ensure that there is no oxygen in the anaerobic chamber. Design the plate layout according to a 96-well template. To run experiments in technical replicates of three, this will allow for 32 different treatments, which is best represented with a four-by-eight grid to set up the vials.
Set up the four-by-eight grid according to the assay plate layout. Then, place 7 milliliter PTFE standard vials in the tray. Vials should only be handled by manipulating the outside of the vial.
Add the exposure medium volume into each vial corresponding to each treatment. To each vial, add the corresponding volume of the chemical variable to be tested according to the plate layout. Now add nitrate to each vial so that the final concentration is 200 micromolar.
Exclude this step for constitutive biosensor treatment blanks.
To add mercury to the vials, first take the four to eight micromolar stock and shake well. Dilute the solution and exposure medium in a 7 milliliter PTFE vial to a concentration of 100 to 250 nanomolar to make a working mercury solution.
From this working solution, add mercury to the required vials according to the plate layout. After adding the mercury or cadmium, manually shake the plate in an orbital motion. The experiment may be paused now depending on the time required for mercury or cadmium to speciate in solution.
When the experiment is resumed, gently pipette biosensor stock back and forth to ensure homogeneity. Then add 100 microliters of the biosensor stock to each vial and manually shake the plate as before. Warm up the plate reader to 37 degrees Celsius and set up a kinetic run for ten hours with reads every two point five to five minutes.
An orbital shaking in between readings, set up the run to take fluorescence measurements with a fluorescence excitation of 440 nanometers and an emission of 500 nanometers. Now pipette 200 microliters from each PTFE vial in the four by eight grid into the corresponding wells of the 96-well plate.
Pipette back and forth five times before transferring each 200 microliters. 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.
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