TITLE: PlatereaderPCAoxidationAssay
AUTHORS: Lev Tsypin, Dianne K Newman, Allen W Chen, Scott Saunders

[DESCRIPTION]
Protocol for measuring phenazine-1-carboxylic acid oxidation in a plate reader

[STEPS]
SECTION: Pre-grow cultures for assay
1. Two days before, streak frozen culture stock on LB agar plate, grow over night at 30 ºC; inoculate liquid cultures for next step with a mixed patch of cells.
SECTION: Pre-grow cultures for assay
2. Grow 5 mL cultures of cells overnight at 30 ºC in LB for 17 hours +/- 10 minutes.
SECTION: Pre-grow cultures for assay
2.1. Depending on the experiment, tubes are either slanted shaking at 250 rpm or standing non-shaking with caps sealed with Parafilm
SECTION: Prepare cells for assay
3. Wash entire volume into basal medium with no terminal electron acceptor and no PCA (essentially just buffer and salts): 20 mM potassium phosphate buffer (pH 7-7.1); 1 mM sodium sulfate; 10 mM ammonium chloride; and 1× freshwater salt solution (17.1 mM sodium chloride, 1.97 mM magnesium chloride, 0.68 mM calcium chloride, and 6.71 mM potassium chloride)
SECTION: Prepare cells for assay
3.1. In 1 mL aliquots, wash 3x into basal medium by spinning for 2 min at 6000x(g), aspirating with vacuum trap, and resuspending by pipetting.
SECTION: Prepare cells for assay
3.2. Measure OD600 and normalize all cultures to OD600 = 0.2-1, target OD600 = 0.5.
SECTION: Prepare cells for assay
3.3. For slow growing cultures, like menAubiC-tlKO, this amounts to spinning down 1 mL of the overnight culture and resuspending it in 250 µL, then adjusting the volume. May need to scale up if inoculating a lot of wells.
SECTION: Prepare cells for assay
4. Bring cultures into the anaerobic chamber.
SECTION: Prepare cells for assay
5. Transfer washed cultures to anoxic microcentrifuge tubes (tubes that have been in chamber for at least three days).
SECTION: Prepare cells for assay
6. Let stand for at least 1 hour. To test that this is fine, can track parallel culture with resazurin to see when it turns pink or clear.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
7. Do this as much as possible while cells are incubating in the anoxic chamber
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
8. Prepare reduced PCA calibration wells
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
8.1. Prepare 500 µM solution by diluting PCAred stock in basal medium.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
8.2. In one row of the plate, prepare calibration (in µM): 250, 200, 175, 150, 125, 100, 75, 50, 25, 10, 5, 0.

This corresponds to the following volumes of the 500 µM PCAred per well (in µL): 100, 80, 70, 60, 50, 40, 30, 20, 10, 4, 2, 0.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
8.3. Bring total volume in each well to 200 µL with basal medium
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
9. Desired concentration of PCA in PCA wells is 200 µM (40 µL of 1 mM stock).
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
10. Desired concentration of cells is 40 µL of OD600 = 0.5 culture for a target of OD600 = 0.1 in the wells.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
11. Other components depend on the experiment: e.g., 2 µL of 1 M sodium nitrate for a 10 mM experimental concentration.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
12. Note: all solutions prepared in the same basal medium.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
13. Final volume in each well is 200 µL.
SECTION: Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
14. Order of preparing wells (except calibration): basal medium, then PCA, then TEA, then cells (always last).
SECTION: Plate reader protocol (BioTek Synergy 4 or HTX)
15. Incubate at 30 ºC throughout experiment. If using HTX model, set the temperature limits +/- 1 ºC to prevent condensation


SECTION: Plate reader protocol (BioTek Synergy 4 or HTX)
16. Medium shaking throughout experiment
SECTION: Plate reader protocol (BioTek Synergy 4 or HTX)
18. Measure every 5 minutes
SECTION: Plate reader protocol (BioTek Synergy 4 or HTX)
17. Measure absorbance at 440 and 600 nm (PCA and cells, respectively), as well as fluorescence 
(360/40 ex and 528/20 em). Test that sensitivity is such that the calibration curve spans the dynamic range (different on each instrument). Include pathlength correction for absorbance measurements.