TITLE: Transcription Factor Binding: Singleplex Assay for Function Measurements
AUTHORS: David Ross

[DESCRIPTION]
This protocol outlines an assay for measuring the function of plasmid variants in singleplex. 

The inputs include separate E. coli glycerol stocks for each of 3 variants, distributed according to the provided function plate map (see attachments). The protocol begins with several growths which convert the separate glycerol stocks into cultures that have reached stationary phase in a 96-well plate. The glycerol stocks are first grown overnight in separate tubes. The next morning, the optical density (OD) of each culture is measured, and then each culture is distributed into a 96-well growth plate. This plate is placed in a plate reader/incubator to grow to stationary phase (~12 hours) without antibiotics or additives (except those required for plasmid maintenance). After this point, the cultures are ready to act as an inputs for the next growth cycles where a 2-fold dilution series of the corresponding inducer is introduced. Throughout the subsequent growths, optical density (OD) and fluorescent measurements are recommended to be taken every 5 minutes and at the end of each growth plate's incubation. The growth cycles are all ~3 hours long, so that cells stay in mid-log phase. At the end of the last growth cycle, the cultures in the growth plate act as input for quantification using either a flow-cytometry or a plate reader. 

Note that unlike the related fitness assays, this assay does not use a selection antibiotic.

[STEPS]
SECTION: Fill in required Metadata
1. 
 

Next, select the step-case below that matches the type of transcription factor variants you are measuring. Below, you will see step cases for each of the transcription factor genes of interest. Please select the gene of interest you are investigating to ensure you follow the protocol with the correct reagents. 

After the third growth plate finishes incubating, you will see another step-case, where you can choose to perform final calibrated Function measurements by using a flow cytometer or plate reader. 

See the Attachments section for suggested plate map layouts for growth plates depending on if you choose flow cytometry or plate reader measurements. The plate reader measurements have one plate map for the variant measurement and a seprarate plate map for the fluorescent/OD calibration measurement.
SECTION: Final Measurements - Select Flow Cytometer or Plate Reader
25. Here we provide a step-case to chose between using flow cytometry or a plate reader to perform the final calibration function measurements using a fluorescent standard.
SECTION: Data Analysis
29. Calculate the quantitative Function by subtracting the mean fluorescent signal of the non-fluorescent control from the measured variants.
SECTION: Data Analysis
28. Using the calibrated data, calculate the mean fluorescence per cell in molecules of equivalent fluorophore (MEF) for each sample, including the non-fluorescent control samples, and the standards. 
SECTION: Plate Reader Fluorescent Measurement
27. Calibrate the OD600 and fluorescence measurements using silica microspheres (950µm) and a fluorescent dye (Sulforhodamine 101). This requires using a new plate, following a fluorescent calibration protocol: The reference for performing this measurement

Note: For additional instruction see References Beal 2018, Beal 2020, and Beal 2022.

SECTION: Plate Reader Fluorescent Measurement
26. Put the third growth plate back into the plate reader (without the gas-permeable seal) and perform a single additional (i.e., end-point) measurement of OD600, OD700 and fluorescence.
SECTION: Data Analysis - Calculating Function
31. For each sample, the quantitative Function is the mean fluorescence for that sample with the background signal subtracted.
SECTION: Data Analysis - Calculating Function
30. Calculate the mean fluorescence signal of the singlet cell events for each sample measured with the (fluorescent) selection plasmid.
SECTION: Data Analysis - Calculating Function
29. Calculate the mean fluorescence signal of the singlet cell events for all measurements of the non-fluorescent control cells. This is the background signal.
SECTION: Flow cytometer preparation &amp; measurements
28. Measure the fluorescence background usin a non-fluorescent control (i.e., E. coli cells of the same strain with a plasmid that is similar to the selection plasmid, but with no fluorescent protein).
SECTION: Flow cytometer preparation &amp; measurements
27. Measure the fluorescence of the cells containing the selection plasmid on a flow cytometer equipped with a 96-well plate autosampler using an excitation wavelength and emission filter appropriate for the fluorescent protein in the plasmid. 
Use fluorescence calibration beads with each batch of samples to facilitate the calibration of flow cytometry data to molecules of equivalent fluorophore. 
Use automated gating to distinguish cell events and singlet cell events from other background events for each sample measured with cytometry, with the same gating strategy applied to fluorescent and non-fluorescent control cells.
SECTION: Flow cytometer preparation &amp; measurements
26. Transfer 5 μl of each cell sample to a well in a round-bottom 96-well plate containing 195 μl of PBS supplemented with 170 μg/mL chloramphenicol. 
Note: Chloramphenicol is added to the PBS to halt production of new proteins in the E. coli cells to give a more stable fluorescence measurement, since it can take over one hour to measure all of the samples. 
SECTION: Third Growth Plate
24. After incubation, remove gas-permeable seal from the third growth plate and perform the quantitative function measurements using one of the following options (Flow Cytometer or Plate Reader).
SECTION: Third Growth Plate
23.1. Measure OD600, OD700, and  fluorescence (569 nm excitation, 593 nm emission for mScarlet-I3 fluorescent protein) every 5 minutes throughout the incubation. 
SECTION: Third Growth Plate
23. Incubate the third growth plate for 3 hours and 5 minutes at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter). 
The OD and fluorescent readings take a total of 5 extra minutes - this is why the incubation in 3 hours and 5 minutes.
SECTION: Third Growth Plate
22. Apply gas-permeable seal to the third growth plate.
SECTION: Third Growth Plate
21. Transfer 50 μL from each well of the second growth plate to corresponding wells of the third growth plate.
SECTION: Second Growth Plate
20. After incubation, remove gas-permeable seal from the second growth plate and measure end-point OD600.

SECTION: Second Growth Plate
19.3. Approximately ten minutes before the end of the 12-hour incubation, pre-warm the third growth plate. Note: Adjust pre-warming temperature and timing so that the media temperature in the plate is 37C at the end of the pre-warming, and so that the pre-warming step ends at the same time as the 12-hour incubation.
SECTION: Second Growth Plate
19.2. Approximately 45 minutes before the end of the 12-hour incubation, prepare the third growth plate. Note: it is similar to the second growth plate, but each well has 450µL of mixed media instead of the previous 490µL.
Create a 2-fold dilution series of IPTG across the columns of the plate using the working media (M9-gly-kan-DMSO) as a dilutor. Note: the exception is that all wells in column one should have no inducer (450 µL of working media only). 
Each well should end with 450µL of mixed media (working media + inducer). 
The highest final concentration of IPTG is 2000µmol/L, which is a 2-fold dilution from the IPTG working solution (4000µmol/L)
Final IPTG concentrations (µmol/L) in columns 1-12 should be:
0, 1.953, 3.908, 7.813, 15.625, 31.25, 62.5, 125, 250,  500, 1000, 2000

SECTION: Second Growth Plate
19.1. Measure OD600, OD700, and  fluorescence (569 nm excitation, 593 nm emission for mScarlet-I3 fluorescent protein)  every 5 minutes throughout the incubation. 
SECTION: Second Growth Plate
19. Incubate the second growth plate for 3 hours and 5 minutes at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
The OD and fluorescent readings take a total of 5 extra minutes - this is why the incubation in 3 hours and 5 minutes.
SECTION: Second Growth Plate
18. Apply gas-permeable seal to the second growth plate.
SECTION: Second Growth Plate
17. Transfer 10 μL from each well of the first growth plate to corresponding wells of the second growth plate.
There are some subtleties in the 96-channel pipetting required to get a reproducible transfer, details are described in the SI of this paper: https://academic.oup.com/synbio/article/7/1/ysac013/6659220 
SECTION: Second Growth Plate
16. After 12-hour incubation, remove gas-permeable seal. 
SECTION: First Growth Plate: cells reach stationary phase
15.2. Approximately ten minutes before the end of the 12-hour incubation, pre-warm the second growth plate. Note: Adjust pre-warming temperature and timing so that the media temperature in the plate is 37C at the end of the pre-warming, and so that the pre-warming step ends at the same time as the 12-hour incubation.
SECTION: First Growth Plate: cells reach stationary phase
15.1. Approximately 45 minutes before the end of the 12-hour incubation, prepare the second growth plate. 
Create a 2-fold dilution series of IPTG across the columns of the plate using the working media (M9-gly-kan-DMSO) as a dilutor. Note: the exception is that all wells in column one should have no inducer (490 µL of working media only). 
Each well should end with 490µL of mixed media (working media + inducer). 
The highest final concentration of IPTG is 2000µmol/L, which is a 2-fold dilution from the IPTG working solution (4000µmol/L)
Final IPTG concentrations (µmol/L) in columns 1-12 should be:
0, 1.953, 3.908, 7.813, 15.625, 31.25, 62.5, 125, 250,  500, 1000, 2000
SECTION: First Growth Plate: cells reach stationary phase
15. Incubate for 12 hours at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
Measure OD600, OD700, and fluorescence (569 nm excitation, 593 nm emission for the mScarlet-I3 fluorescent protein) every 5 minutes throughout the incubation. 
SECTION: First Growth Plate: cells reach stationary phase
14. Apply gas-permeable seal to the first growth plate.
SECTION: First Growth Plate: cells reach stationary phase
13.2. Add 50 μL cell culture into the appropriate wells.
SECTION: First Growth Plate: cells reach stationary phase
13.1. Add 450 μL of the final working media (M9-gly-kan-DSMO) to the appropriate wells.
SECTION: First Growth Plate: cells reach stationary phase
13. Prepare first growth plate: 
SECTION: Prepare the automation system or liquid handler
12. Load in the bacterial cultures in 14 mL snap-cap culture tubes (with lids removed).
SECTION: Prepare the automation system or liquid handler
11. Load in the first growth plate (Growth Plate 1).
SECTION: Prepare the automation system or liquid handler
10.2. For downstream measurements performed by flow-cytometry, fill column 5 with focusing fluid and column 6 with PBS.
SECTION: Prepare the automation system or liquid handler
10.1. Fill column 1 of the 6-reservoir plate with the IPTG inducer working solution.
SECTION: Prepare the automation system or liquid handler
10. Load a 6-reservoir plate with a lid.
SECTION: Prepare the automation system or liquid handler
9. Load a single cavity waste reservoir without lid, filled with ~100 mL of tap water
SECTION: Prepare the automation system or liquid handler
8. Load a single cavity media reservoir with a lid, filled with the remaining M9-gly-kan-DMSO final working media.
SECTION: Prepare media and inducer working solutions
7.3. Mix the inducer working solution well.
SECTION: Prepare media and inducer working solutions
7.2. Add 80 µL IPTG stock (see Materials: stock at 1 mol/L in water) to Falcon tube
SECTION: Prepare media and inducer working solutions
7.1. Transfer 19.92 mL M9-gly-kan-DMSO media to a 50mL Falcon tube
SECTION: Prepare media and inducer working solutions
7. Prepare the inducer working solution: 4 mmol/L IPTG in M9-gly-kan-DMSO
SECTION: Prepare media and inducer working solutions
6.4. Mix the media bottle well
SECTION: Prepare media and inducer working solutions
6.3. Add 1.256 mL DMSO to the media bottle, creating M9-gly-kan-DMSO at 0.5% DMSO
SECTION: Prepare media and inducer working solutions
6.2. Add 250 µL kanamycin (kan) to the media bottle, creating M9-gly-kan at 50 µg/mL kanamycin.
SECTION: Prepare media and inducer working solutions
6.1. Add 250 mL M9-gly to a sterile media bottle.
SECTION: Prepare media and inducer working solutions
6. Prepare the final working media: M9-gly-kan-DMSO
SECTION: Quality control the overnight culture
5. After incubation, measure the OD600 of each overnight culture as a growth check.
SECTION: Culture Preparation &amp; Overnight Growth
4. Incubate cultures overnight (1440 min) at 37°C with shaking  at 300 rpm.

SECTION: Culture Preparation &amp; Overnight Growth
3. For each of the variants to be tested, fill a 15 mL snap-cap culture tube with 5 mL of M9-gly-kan media.
Use a scraping from the glycerol stock for each clonal variant and place into its culture tube.
Each run should contain a non-fluorescent variant as a background control.
SECTION: Culture Preparation &amp; Overnight Growth
2. Mix 30 mL M9-gly media with 30 µL of kanamycin stock (50 mg/mL) to get working media with 50 µg/mL kanamycin, M9-gly-kan.
SECTION: Final Measurements - Select Flow Cytometer or Plate Reader
26. Here we provide a step-case to chose between using flow cytometry or a plate reader to perform the final calibration function measurements using a fluorescent standard.
SECTION: Data Analysis
30. Calculate the quantitative Function by subtracting the mean fluorescent signal of the non-fluorescent control from the measured variants.
SECTION: Data Analysis
29. Using the calibrated data, calculate the mean fluorescence per cell in molecules of equivalent fluorophore (MEF) for each sample, including the non-fluorescent control samples, and the standards. 
SECTION: Plate Reader Fluorescent Measurement
28. Calibrate the OD600 and fluorescence measurements using silica microspheres (950µm) and a fluorescent dye (Sulforhodamine 101). This requires using a new plate, following a fluorescent calibration protocol: The reference for performing this measurement

Note: For additional instruction see References Beal 2018, Beal 2020, and Beal 2022.

SECTION: Plate Reader Fluorescent Measurement
27. Put the third growth plate back into the plate reader (without the gas-permeable seal) and perform a single additional (i.e., end-point) measurement of OD600, OD700 and fluorescence.
SECTION: Data Analysis - Calculating Function
32. For each sample, the quantitative Function is the mean fluorescence for that sample with the background signal subtracted.
SECTION: Data Analysis - Calculating Function
31. Calculate the mean fluorescence signal of the singlet cell events for each sample measured with the (fluorescent) selection plasmid.
SECTION: Data Analysis - Calculating Function
30. Calculate the mean fluorescence signal of the singlet cell events for all measurements of the non-fluorescent control cells. This is the background signal.
SECTION: Flow cytometer preparation &amp; measurements
29. Measure the fluorescence background using a non-fluorescent control (i.e., E. coli cells of the same strain with a plasmid that is similar to the selection plasmid, but with no fluorescent protein).
SECTION: Flow cytometer preparation &amp; measurements
28. Measure the fluorescence of the cells containing the selection plasmid on a flow cytometer equipped with a 96-well plate autosampler using an excitation wavelength and emission filter appropriate for the fluorescent protein in the plasmid. 
Use fluorescence calibration beads with each batch of samples to facilitate the calibration of flow cytometry data to molecules of equivalent fluorophore. 
Use automated gating to distinguish cell events and singlet cell events from other background events for each sample measured with cytometry, with the same gating strategy applied to fluorescent and non-fluorescent control cells.
SECTION: Flow cytometer preparation &amp; measurements
27. Transfer 5 μl of each cell sample to a well in a round-bottom 96-well plate containing 195 μl of PBS supplemented with 170 μg/mL chloramphenicol. 
Note: Chloramphenicol is added to the PBS to halt production of new proteins in the E. coli cells to give a more stable fluorescence measurement, since it can take over one hour to measure all of the samples. 
SECTION: Third Growth Plate
25. After incubation, remove gas-permeable seal from the third growth plate and perform the quantitative function measurements using one of the following options (Flow Cytometer or Plate Reader).
SECTION: Third Growth Plate
24.1. Measure OD600, OD700, and  fluorescence (569 nm excitation, 593 nm emission for mScarlet-I3 fluorescent protein) every 5 minutes throughout the incubation. 
SECTION: Third Growth Plate
24. Incubate the third growth plate for 3 hours and 5 minutes at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter). 
The OD and fluorescent readings take a total of 5 extra minutes - this is why the incubation in 3 hours and 5 minutes.
SECTION: Third Growth Plate
23. Apply gas-permeable seal to the third growth plate.
SECTION: Third Growth Plate
22. Transfer 50 μL from each well of the second growth plate to corresponding wells of the third growth plate.
SECTION: Second Growth Plate
21. After incubation, remove gas-permeable seal from the second growth plate and measure end-point OD600.

SECTION: Second Growth Plate
20.3. Approximately ten minutes before the end of the 12-hour incubation, pre-warm the third growth plate. Note: Adjust pre-warming temperature and timing so that the media temperature in the plate is 37C at the end of the pre-warming, and so that the pre-warming step ends at the same time as the 12-hour incubation.
SECTION: Second Growth Plate
20.2. Approximately 45 minutes before the end of the 12-hour incubation, prepare the third growth plate. Note: it is similar to the second growth plate, but each well has 450µL of mixed media instead of the previous 490µL.
Create a 2-fold dilution series of 1S-TIQ across the columns of the plate using the working media (M9-gly-kan-DMSO) as a dilutor. Note: the exception is that all wells in column one should have no inducer (450 µL of working media only). 
Each well should end with 450µL of mixed media (working media + inducer). 
The highest final concentration of 1S-TIQ is 250µmol/L which is a 2-fold dilution from the 1S-TIQ working solution (500µmol/L)
Final 1S-TIQ concentrations (µmol/L) in columns 1-12 should be:
0, 0.245, 0.488, 0.978, 1.953, 3.908, 7.813, 15.625, 31.25, 62.5, 125, and 250

SECTION: Second Growth Plate
20.1. Measure OD600, OD700, and  fluorescence (569 nm excitation, 593 nm emission for mScarlet-I3 fluorescent protein)  every 5 minutes throughout the incubation. 
SECTION: Second Growth Plate
20. Incubate the second growth plate for 3 hours and 5 minutes at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
The OD and fluorescent readings take a total of 5 extra minutes - this is why the incubation in 3 hours and 5 minutes.
SECTION: Second Growth Plate
19. Apply gas-permeable seal to the second growth plate.
SECTION: Second Growth Plate
18. Transfer 10 μL from each well of the first growth plate to corresponding wells of the second growth plate.
There are some subtleties in the 96-channel pipetting required to get a reproducible transfer, details are described in the SI of this paper: https://academic.oup.com/synbio/article/7/1/ysac013/6659220 
SECTION: Second Growth Plate
17. After 12-hour incubation, remove gas-permeable seal. 
SECTION: First Growth Plate: cells reach stationary phase
16.2. Approximately ten minutes before the end of the 12-hour incubation, pre-warm the second growth plate. Note: Adjust pre-warming temperature and timing so that the media temperature in the plate is 37C at the end of the pre-warming, and so that the pre-warming step ends at the same time as the 12-hour incubation.
SECTION: First Growth Plate: cells reach stationary phase
16.1. Approximately 45 minutes before the end of the 12-hour incubation, prepare the second growth plate. 
Create a 2-fold dilution series of 1S-TIQ across the columns of the plate using the working media (M9-gly-kan-DMSO) as a dilutor. Note: the exception is that all wells in column one should have no inducer (490 µL of working media only). 
Each well should end with 490µL of mixed media (working media + inducer). 
The highest final concentration of 1S-TIQ is 250µmol/L which is a 2-fold dilution from the 1S-TIQ working solution (500µmol/L)
Final 1S-TIQ concentrations (µmol/L) in columns 1-12 should be:
0, 0.245, 0.488, 0.978, 1.953, 3.908, 7.813, 15.625, 31.25, 62.5, 125, and 250
SECTION: First Growth Plate: cells reach stationary phase
16. Incubate for 12 hours at 37°C, with fastest shaking possible in the plate reader (e.g., in Biotek Neo2SM reader: double orbital shaking at 807 cpm and 1 mm shaking diameter).
Measure OD600, OD700, and fluorescence (569 nm excitation, 593 nm emission for the mScarlet-I3 fluorescent protein) every 5 minutes throughout the incubation. 
SECTION: First Growth Plate: cells reach stationary phase
15. Apply gas-permeable seal to the first growth plate.
SECTION: First Growth Plate: cells reach stationary phase
14.2. Add 50 μL cell culture into the appropriate wells.
SECTION: First Growth Plate: cells reach stationary phase
14.1. Add 450 μL of the final working media (M9-gly-kan-DSMO) to the appropriate wells.
SECTION: First Growth Plate: cells reach stationary phase
14. Prepare first growth plate: 
SECTION: Prepare the automation system or liquid handler
13. Load in the bacterial cultures in 14 mL snap-cap culture tubes (with caps removed).
SECTION: Prepare the automation system or liquid handler
12. Load in the first growth plate (Growth Plate 1).
SECTION: Prepare the automation system or liquid handler
11.2. For downstream measurements performed by flow-cytometry, load 5 mL to 10 mL focusing fluid into column 5, and load 45 mL PBS with 170 ug/mL chloramphenicol into column 6.
SECTION: Prepare the automation system or liquid handler
11.1. Fill column 1 of the 6-reservoir plate with the 1S-TIQ inducer working solution.
SECTION: Prepare the automation system or liquid handler
11. Load a 6-reservoir plate with a lid.
SECTION: Prepare the automation system or liquid handler
10. Load a single cavity waste reservoir without lid, filled with ~100 mL of tap water
SECTION: Prepare the automation system or liquid handler
9. Load a single cavity media reservoir with a lid, filled with the remaining M9-gly-kan-DMSO final working media.
SECTION: Prepare media and inducer working solutions
8.2. Mix the final working media well.
SECTION: Prepare media and inducer working solutions
8.1. Add 1.156 mL DMSO to the initial working media bottle to create the final working media of M9-gly-kan-DMSO.
Note: This creates a final working media with 0.5% DMSO.
SECTION: Prepare media and inducer working solutions
8. Create the final working media: M9-gly-kan-DMSO
SECTION: Prepare media and inducer working solutions
7.3. Mix the inducer working solution well.
SECTION: Prepare media and inducer working solutions
7.2. Add 100 µL of the 1S-TIQ stock (stock at 0.1 mol/L in DMSO) to the working media in the Flacon tube. 
Note: Since the 1S-TIQ stock is already in DSMO, this step results in 500 µmol/L 1S-TIQ in 0.5% DMSO
SECTION: Prepare media and inducer working solutions
7.1. Transfer 19.9 mL M9-gly-kan from the 250 mL initial working media container to a 50 mL Falcon tube.
SECTION: Prepare media and inducer working solutions
7. Prepare the inducer working solution: 500 µmol/L 1S-TIQ 0.5% DMSO in M9-gly-kan
SECTION: Prepare media and inducer working solutions
6.3. Mix the initial working media well
SECTION: Prepare media and inducer working solutions
6.2. Add 250 µL kanamycin (kan) to the media to get an initial working media with 50 µg/mL kanamycin, M9-gly-kan.
SECTION: Prepare media and inducer working solutions
6.1. Add 250 mL M9-gly to a sterile media bottle.
SECTION: Prepare media and inducer working solutions
6. Prepare the initial working media: M9-gly-kan
SECTION: Quality control the overnight culture
5. After incubation, measure the OD600 of each overnight culture as a growth check.
SECTION: Culture Preparation &amp; Overnight Growth
4. Incubate cultures overnight (1440 min) at 37°C with shaking  at 300 rpm.

SECTION: Culture Preparation &amp; Overnight Growth
3. For each of the variants to be tested, fill a 15 mL snap-cap culture tube with 5 mL of M9-gly-kan media.
Use a scraping from the glycerol stock for each clonal variant and place into its culture tube.
Each run should contain a non-fluorescent variant as a background control.
SECTION: Culture Preparation &amp; Overnight Growth
2. Mix 30 mL M9-gly media with 30 µL of kanamycin stock (50 mg/mL) to get working media with 50 µg/mL kanamycin, M9-gly-kan.