Jan 17, 2020
High-Throughput Beta-glucuronidase (GUS) assay for Phaeodactylum tricornutum
- 1J. Craig Venter Institute;
- 2J. Craig Venter Institute, Synthetic Biology & Bioenergy Group
- Protist Research to Optimize Tools in Genetics (PROT-G)
- JCVI West Protocols
Protocol Citation: Erin Garza, Vincent A Bielinski 2020. High-Throughput Beta-glucuronidase (GUS) assay for Phaeodactylum tricornutum. protocols.io https://dx.doi.org/10.17504/protocols.io.bbexijfn
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: January 15, 2020
Last Modified: January 17, 2020
Protocol Integer ID: 31927
Keywords: Gus assay, diatoms, Phaeodactylum tricornutum, high-throughput
Abstract
A high-throughput method for measuring β-glucuronidase (GUS) activity in the diatom Phaeodactylum tricornutum. This protocol has been optimized for 250 μl volumes. For larger volumes see the following protocol dx.doi.org/10.17504/protocols.io.hefb3bn, which this protocol was based off of.
Materials
MATERIALS
MUGGold BiotechnologyCatalog #MUG
Sodium carbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #222321
B-PER™ Bacterial Protein Extraction ReagentThermo FisherCatalog #78243
Flat bottom transparent and opaque 96-well plates
GUS extraction buffer- 50 mM NaPO4H2 (pH 7), 0.1% Triton X-100, and 10 µM βME + 1 mM 4-Methylumbelliferyl β-D-Glucuronide (MUG)
GUS stop buffer- 0.2 M Na2CO3
Plate reader
Swing bucket centrifuge with plate adapter
Before start
Phaeodactylum tricornutum cultures were initially grown in 5 ml L1 + antibiotics in a 50-ml conical at 18°C until the cell concentration reached at least 1x106 cells ml-1.
Centrifuge
Centrifuge
Transfer 250 μl of each P. tricornutum culture to a 96-well plate and centrifuge at 3000 x g for 10 min. Discard supernatant.
Lyse
Lyse
To lyse the cells, add 150 µl bacterial protein extraction reagent (B-PER, ThermoFisher) to each well and mix by pipetting.
Centrifuge
Centrifuge
Centrifuge plate for 10 min at 3000 x g. Transfer supernatants to a new 96-well plate, being careful not to disturb the cell debris.
Extract
Extract
Transfer 50 µl of each lysate to a new plate and add 125 µl GUS extraction buffer + 1 mM MUG to each well. Incubate the plate for 1 h at 37°C.
GUS extraction buffer= 50 mM NaPO4H2 (pH 7), 0.1% Triton X-100, and 10 µM βME
Stop Reaction
Stop Reaction
To stop the reaction, add 150 µl GUS stop buffer (0.2 M Na2CO3) to each well and mix by pipetting.
Transfer 200 µl quenched reaction to an opaque 96-well plate.
Read fluorescence
Read fluorescence
Determine fluorescence using a plate reader. Settings: excitation- 360 nm; emission- 440 nm.
If fluorescence readings are too high to get a readout, dilute with additional stop buffer.
Normalization
Normalization
Use remaining cell lysates to perform a BCA assay (or an equivalent assay) to normalize the GUS activity to total cell protein for each culture.