Reporter Expression Detection

Similar to when creating a standard curve potentiostatically the working electrode must be held at a specific oxidation potential for the detection of that analyte. Testing for this requires a culture of cells that will be able to report through a hydrolase enzyme. To perform the detection a three electrode setup will be needed.

• Grow cells overnight in 3mL of LB
• Pellet cells at 3750rpm for 10 minutes
• Remove supernatant
• Resuspend in 1mL of 0.1M pH7 PBS
• Add to 25mL 0.1M pH7 PBS in an electrochemical cell
• Add the solution to be tested for activation of the reporter gene
• Add the electrodes and close the cell
• Insert needle and bubble nitrogen or argon gas into the solution for 5 minutes
• Hold the working electrode at the oxidation potential for the analyte product
• Wait 5 minutes for current stabilization
• Add the sugar-analyte substrate
• Observe any current changes over a time period
• Relate to concentration of analyte produced using a standard curve