I have been working on genetically engineering an E. coli strain to autotrophically oxidize arsenite into arsenate for bioremediation of arsenic contamination in groundwater. For my research, I have been finding ways to quantitatively measure how much arsenite the bacteria oxidizes over time. After researching ways to quantitatively measure arsenite being oxidized, I have concluded to use the method of Johnson and Pilson (https://www.sciencedirect.com/science/article/pii/S0003267072800059?via%3Dihub).
The arsenate that is formed from arsenite oxidation forms a molybdenum-blue complex with phosphate in the water. As the concentration of arsenate increases, the absorbance value of the water should increase proportionally.
Here was my plan: First, after growing my transgenic bacteria on an amp-plate, I would pipette out various concentrations of cells and arsenite in wells of a 96-well spot plate. I currently plan to use the Fisher Scientific Multiskan FC microplate photometer. I planned on incubating the cells while shaking at 37 C. The main problem I have come across is that my experiment would not be controlled. Over time, the growth of cells will change the absorbance value, not just the molybdenum-blue complex. I need to isolate the variable of just arsenite oxidation.
I have thought of a few possible solutions, but I feel as if I may be missing something crucial. When learning about microplate readers, I know that researchers can use them to measure gene expression. How do these researchers eliminate the variable of cell growth effects on absorbance values?
For one, I have thought of finding a way to filter the cells to one side of the well where they will not affect the absorbance values. Would it be possible for me to cut out a dialysis bag to where it is dipping into the well? In that case, the cells could be suspended in the well, oxidizing arsenite (if my hypothesis is correct).
Anyway, I really want to find a simple method for quantitatively measuring arsenite concentrations over time using a microplate reader without having cell growth affecting absorbance values.