TITLE: NanoLuciferase Assay 
AUTHORS: afedoriouk, Morgan Farrell, Nicholas J Shikuma

[DESCRIPTION]
Protocol for a luciferase assay to quantify promoter expression in marine bacteria.

[STEPS]
SECTION: Day 1: Streak out strains
1. 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

    
SECTION: Day 2: Create Sub-Culture
2. 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
SECTION: Day 3: Inoculate Experimental Cultures
8. Inoculate experimental culture from the sub-culture created in Day 2 with 1:100 dilution of media to sub-culture.

SECTION: Day 3: Inoculate Experimental Cultures
9. 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.
SECTION: Day 3: Inoculate Experimental Cultures
10. Take optical density OD600 measurement right after inoculation to have a time point 0 (T0).
SECTION: Day 3: Inoculate Experimental Cultures
11. 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
SECTION: Day 3: Performing NanoLuciferase Assay
13. 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.
SECTION: Day 3: Performing NanoLuciferase Assay
12. Prepare mastermix for blanks reactions.   
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.

 

SECTION: Day 3: Performing NanoLuciferase Assay
14. 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
SECTION: Day 3: Performing NanoLuciferase Assay
15. 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.
SECTION: Day 3: Performing NanoLuciferase Assay
16. 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
SECTION: Day 3: Performing NanoLuciferase Assay
17. Pipette 20 µL of mastermix in each well first.
SECTION: Day 3: Performing NanoLuciferase Assay
18. Then, pipette 20 µL of sample into luciferase plate with mastermix using a multichannel pipette.

 

SECTION: Day 3: Performing NanoLuciferase Assay
19. 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.
SECTION: Day 2: Create Sub-Culture
3. Incubate at 25ºC shaking at 200 rpm for 24 hours.
 

SECTION: Biofilm Only + 1 Day: Spot Cultures for Biofilm
4. Take the optical density (OD600) measurement of the sub-cultures with a spectrophotometer to ensure they are in stationary phase.
SECTION: Biofilm Only + 1 Day: Spot Cultures for Biofilm
5. 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
SECTION: Biofilm Only + 1 Day: Spot Cultures for Biofilm
6. Resuspend bacterial pellet with 75 µL of liquid media.
SECTION: Biofilm Only + 1 Day: Spot Cultures for Biofilm
7. Spot the entire 75 µL onto a single agar plate of the appropriate media and antibiotics.
Inoculate plate for 24 hours at 25ºC