Nov 14, 2023
PlatereaderPCAoxidationAssay
- Lev Tsypin1,
- Dianne K Newman2,
- Allen W Chen2,
- Scott Saunders3
- 1Stanford University;
- 2California Institute of Technology;
- 3UT Southwestern
Protocol Citation: Lev Tsypin, Dianne K Newman, Allen W Chen, Scott Saunders 2023. PlatereaderPCAoxidationAssay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xm6dlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 14, 2023
Last Modified: November 14, 2023
Protocol Integer ID: 90896
Funders Acknowledgement:
NIH
Grant ID: 1R01AI127850-01A1
Doren Family Foundation
NSF GRFP
Abstract
Protocol for measuring phenazine-1-carboxylic acid oxidation in a plate reader
Pre-grow cultures for assay
Pre-grow cultures for assay
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.
Grow 5 mL cultures of cells overnight at 30 ºC in LB for 17 hours +/- 10 minutes.
Depending on the experiment, tubes are either slanted shaking at 250 rpm or standing non-shaking with caps sealed with Parafilm
Prepare cells for assay
Prepare cells for assay
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)
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.
Measure OD600 and normalize all cultures to OD600 = 0.2-1, target OD600 = 0.5.
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.
Bring cultures into the anaerobic chamber.
Transfer washed cultures to anoxic microcentrifuge tubes (tubes that have been in chamber for at least three days).
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.
Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
Set up plate (BRAND PureGradeTM S, Cat. No. 781671) inside anoxic chamber
Do this as much as possible while cells are incubating in the anoxic chamber
Prepare reduced PCA calibration wells
Prepare 500 µM solution by diluting PCAred stock in basal medium.
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.
Bring total volume in each well to 200 µL with basal medium
Desired concentration of PCA in PCA wells is 200 µM (40 µL of 1 mM stock).
Desired concentration of cells is 40 µL of OD600 = 0.5 culture for a target of OD600 = 0.1 in the wells.
Other components depend on the experiment: e.g., 2 µL of 1 M sodium nitrate for a 10 mM experimental concentration.
Note: all solutions prepared in the same basal medium.
Final volume in each well is 200 µL.
Order of preparing wells (except calibration): basal medium, then PCA, then TEA, then cells (always last).
Plate reader protocol (BioTek Synergy 4 or HTX)
Plate reader protocol (BioTek Synergy 4 or HTX)
Incubate at 30 ºC throughout experiment. If using HTX model, set the temperature limits +/- 1 ºC to prevent condensation
Medium shaking throughout experiment
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.
Measure every 5 minutes