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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.

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