Jan 30, 2023

Public workspaceNanoLuciferase Assay

This protocol is a draft, published without a DOI.
  • 1San Diego State University
Morgan Farrell
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Protocol Citation: afedoriouk, Morgan Farrell, Nicholas J Shikuma 2023. NanoLuciferase Assay . protocols.io https://protocols.io/view/nanoluciferase-assay-cig2ubye
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: October 27, 2022
Last Modified: January 30, 2023
Protocol Integer ID: 71930
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: GBMF9344
National Science Foundation
Grant ID: 1942251
Office of Naval Research
Grant ID: N00014-20-1-2120
Abstract
Protocol for a luciferase assay to quantify promoter expression in marine bacteria.
Materials
ReagentNano-Glo live Cell Assay SystemPromegaCatalog #N2011

Protocol materials
ReagentNano-Glo live Cell Assay SystemPromegaCatalog #N2011
Materials
ReagentNano-Go Live Cell Assay SystemPromegaCatalog #N2011
Step 12
Day 1: Streak out strains
Day 1: Streak out strains
30m
30m
Streak out all strains from frozen stock onto the appropriate antibiotic plates. Including a positive and negative control.
  • Pseudoalteromonas luteoviolacea expressing kanamycin resistant backbone was struck onto NSWT with 300 µg/mL of kanamycin
  • Pseudoalteromonas sp. PS5 expressing kanamycin resistant plamid was struck onto Marine Broth (MB) with 300 µg/mL of kanamycin
  • Wild type marine bacteria were grown on MB only media

30m
Day 2: Create Sub-Culture
Day 2: Create Sub-Culture
Create sub-culture by inoculating 5 colonies from the plate into a 5 mL tube with appropriate media and antibiotics. Repeat for each strain into a separate 5 mL tube.
  • Biological replicates should be in separate sub-cultures
1h
Incubate at 25ºC shaking at 200 rpm for 24 hours.
Note
Plan ahead! Start this step at a time that works best for you to perform the experiments on day 3.

Biofilm Only + 1 Day: Spot Cultures for Biofilm
Biofilm Only + 1 Day: Spot Cultures for Biofilm
Take the optical density (OD600) measurement of the sub-cultures with a spectrophotometer to ensure they are in stationary phase.
10m
Take 1.5ml of sub-culture and transfer into a micro centrifuge tube. Spin down to concentrate bacteria.
  • Spin cultures at 4000g for 10 minutes
  • Remove supernatant and avoid cell pellet
15m
Resuspend bacterial pellet with 75 µL of liquid media.
10m
Spot the entire 75 µL onto a single agar plate of the appropriate media and antibiotics.
  • Inoculate plate for 24 hours at 25ºC
1d
Day 3: Inoculate Experimental Cultures
Day 3: Inoculate Experimental Cultures
Inoculate experimental culture from the sub-culture created in Day 2 with 1:100 dilution of media to sub-culture.

30m
In 125 mL flask add 25 mL of media and 250 µL of sub-culture. Repeat for each strain and biological replicate into its own flask.
5m
Take optical density OD600 measurement right after inoculation to have a time point 0 (T0).
5m
Continue to take optical density measurements to determine the major growth phases that will be tested in the NanoLuciferase assay. 
  • Test 2-3 strains to determine where the OD600 is at. May need to retest every 30 minutes in the beginning to capture exponential phase.
  • To measure OD600:  Fill cuvettes in with 1 mL of sample and flick each vial before taking OD600 measurement
8h
Day 3: Performing NanoLuciferase Assay
Day 3: Performing NanoLuciferase Assay
2h 5m
2h 5m
Prepare mastermix for blanks reactions. ReagentNano-Go Live Cell Assay SystemPromegaCatalog #N2011
  • 4 blanks
  • 2.5 µL of Buffer x 4= 10 µL of Buffer + 0.5 µL of Substrate
  • 17.5 µL of water x 4 = 70 µL 
  • 20 µL of blank media per well

Run the plate with the blanks making sure to label to results as blanks in the computer software.

Note
Always have the machine read the whole plate and not just selected cells that have samples.

10m
Prepare luciferase plate by loading blank samples first to see if any of the wells including the blank are showing signal before the start of the assay.
20m
Prepare the sample only plate in a regular 96-well PCR plate.
  • 100 µL of each sample culture laid out the same as the luciferase plate.
  • Keep the layout the same so you can use a multichannel pipette to transfer the samples from the PCR plate to the luciferase plate. 
  • Shake or mix each flask before pipetting out the sample so that it is evenly mixed
20m
Dilute the samples when making the samples plate. 
  • Past assays have been successful with the following dilution scheme.
  • Pseudoalteromonas luteoviolacea was diluted in a range of 1:10 to 1:100.
  • Pseudoalteromonas sp.PS5 was diluted in a range of 1:1,000 to 1:100,000.

Late stationary and biofilm growth phases showed higher expression and required larger dilutions. Additionally, constitutive promoters required larger dilutions than native promoters.
  • Use serial dilution of 1:100 in a regular 96-well plate first then proceed to do 10-fold dilutions to get to the above dilution levels.
30m
Prepare the luciferase experimental plate. Use the mastermix reaction template to calculate the amount for your number of samples. 
  • i.e. For 72 reactions prep 80 reactions for mastermix
  • 80 x 2.5 µL Buffer/Substrate = 200 µL Buffer + 10 µL Substrate
  • 80 x 17.5 µL MilliQ Water = 1400µL MQ  
  • 20 µL of mastermix per well
5m
Pipette 20 µL of mastermix in each well first.
20m
Then, pipette 20 µL of sample into luciferase plate with mastermix using a multichannel pipette.

Note
Needs to be done as quickly as possible ~1 minute is ideal.

5m
At each time point that you run the assay measure the optical density to normalize your luciferase data by OD600. 
  • Fill cuvettes in with 1 mL of sample. 
  • Flick each vial to mix before taking OD600 measurement.
15m