Oct 15, 2024
Version 3
Transcription Factor Binding: Singleplex Assay for Function Measurements V.3
Version 1 is forked from Singleplex Assay for Function Measurements
- 1NIST
Protocol Citation: David Ross 2024. Transcription Factor Binding: Singleplex Assay for Function Measurements. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzq5qgpk/v3Version created by Open Datasets Initiative
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: August 02, 2024
Last Modified: October 15, 2024
Protocol Integer ID: 109909
Keywords: Deep mutational scanning, protein sequence-function relationships, fitness landscape, laboratory automation, flow cytometry
Disclaimer
The protocol outlined in this document was created as a part of the Pooled, Growth-Based Assays for Protein Function Measurements pipeline for Align to Innovate’s Open Dataset Initiative. Align to Innovate is a non-profit research organization operating under open science principles with the goal of improving science research with programmable experiments. The Open Datasets Initiative is working to accelerate community-driven science with the use of automated labs to pioneer robust data collection methods and curated, high-fidelity, public biological datasets amenable to machine learning. This work was supported by Align to Innovate’s Open Datasets Initiative which receives philanthropic funding in part from Griffin Catalyst. This protocol was typeset by Dana Cortade and David Ross.
Abstract
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.
Materials
Starting cultures:
- a glycerol stock of each of the variants to be tested
- a glycerol stock of a non-fluorescent 'blank' plasmid for background control
Reagents:
- 250 mL of media, M9 with glycerol (M9-gly)
- Kanamycin (kan) stock, 250 uL, 50 mg/mL in water
- DMSO, anhydrous (ThermoFisher, part no. D12345)
- Tap water
- Inducer stock, use either 1 or 2 depending on which transcription factor is being measured:
- For LacI plasmids, use IPTG stock at 120 uL, 1 mol/L in water (pre-prepared ~0.5 mL aliquots at 1 mol/L in water stored in the freezer).
- For RamR plasmids, use 1S-TIQ stock at 40 uL, 0.1 mol/L in DMSO (Ambeed, Part no. A236973) (pre-prepared ~0.5 mL aliquots at 0.1 mol/L in DMSO stored in the freezer).
For plate reader measurements, materials needed to calibrate plate reader FL/OD to molecules of equivalent fluorophore including:
- Phosphate Buffered Saline (PBS) (MilliporeSigma 806544)
- Sterile Water
- 950nm Silica microspheres
For flow cytometry measurements:
- Chloramphenicol (Fisher Scientific BP904-100)
- 1x phosphate-buffered saline (PBS) with 170 ug/mL chloramphenicol
- Fluorescent calibration beads for flow cytometry (Spherotech RCP-30-20A)
Consumables:
- One 50 mL falcon tube (ThermoFisher 339652)
- One 15 mL snap cap tube per variant being tested (Corning 352059)
- Two single-cavity reservoir plates (Agilent 204093-100)
- One sterile media bottle (ThermoFisher 10340001)
- One 6-column reservoir plate (Agilent 204284-100)
- Three 96-well growth plates (Agilent 204799-100)
- Three gas permeable seals (Azenta P98-712)
- For flow cytometry calibration: one 96-well plate(Fisher Scientific 08-772-54)
- For plate reader calibration: one 96-well growth plate (Agilent 204799-100)
Note
To edit these fields, click on the edit symbol (a small square with a pencil in it) located to the right of this step's boarder. Enter the experiment ID generated when the Experiment Requester (or Lead) filled out the Experiment Registration excel. If samples generated using this protocol are used as inputs samples in another experiment/protocol, use the same Experiment ID throughout the pipeline. The operator is the person carrying out the experiment. If several operators will work on the same protocol, enter in their names and indicate which sections of steps each operator will carry out.
Experiment ID:
Operator Name:
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.
LacI control variantsFrom 45 to 47 steps
Follow these steps for testing LacI control variants. You will need IPTG as an inducer.
Culture Preparation & Overnight Growth
Culture Preparation & Overnight Growth
In a 50 mL falcon tube 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.
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 'blank plasmid' non-fluorescent variant as a background control.
Incubate cultures overnight (Overnight ) at 37°C with shaking at 300 rpm.
Quality control the overnight culture
Quality control the overnight culture
After incubation, measure the OD600 of each overnight culture as a growth check.
Prepare working media and inducer working solutions
Prepare working media and inducer working solutions
Prepare the final working media: M9-gly-kan-DMSO
Add 250 mL M9-gly to a sterile media bottle.
Add 250 µL kanamycin (kan at 50mg/mL) to the media bottle, creating M9-gly-kan at 50 µg/mL kanamycin.
Add 1.256 mL DMSO to the media bottle, creating M9-gly-kan-DMSO at 0.5% DMSO
Mix the media bottle well
Prepare the inducer working solution: 4 mmol/L IPTG in M9-gly-kan-DMSO
Transfer 19.92 mL M9-gly-kan-DMSO media to a 50mL Falcon tube
Add 80 µL IPTG stock (see Materials: stock at 1 mol/L in water) to Falcon tube
Mix the inducer working solution well.
Prepare the automation system or liquid handler
Prepare the automation system or liquid handler
Load a single cavity media reservoir with a lid, filled with the remaining M9-gly-kan-DMSO final working media (~231 mL).
Load a another single cavity waste reservoir without lid, filled with ~100 mL of tap water
Load a 6 column reservoir plate with a lid.
Fill column 1 of the 6-reservoir plate with the IPTG inducer working solution.
- If using a plate reader for final measurements: fill column 5 with sterile water and column 6 with PBS.
- If using flow cytometry for final measurements: fill column 5 with focusing fluid and column 6 with PBS.
Load in the first growth plate (Growth Plate 1).
Load in the three LacI variant cultures and blank plasmid culture in 15 mL snap-cap culture tubes (with lids removed).
First Growth Plate: cells reach stationary phase
First Growth Plate: cells reach stationary phase
Prepare first growth plate
- Note: the first growth plate only includes working media and the E.coli cells (no inducer is added).
Add 450 μL of the final working media (M9-gly-kan-DSMO) to the appropriate wells.
Add 50 μL cell culture into the appropriate wells.
Apply gas-permeable seal to the first growth plate.
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.
Approximately 45 minutes before the end of the 12-hour incubation, prepare the second growth plate.
- Create a 2-fold dilution series of inducer working solution (IPTG in working media) across the columns of the plate using the working media 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
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.
Second Growth Plate
Second Growth Plate
After 12-hour incubation, remove gas-permeable seal.
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
Apply gas-permeable seal to the second growth plate.
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.
Measure OD600, OD700, and fluorescence (569 nm excitation, 593 nm emission for mScarlet-I3 fluorescent protein) every 5 minutes throughout the incubation.
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
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.
After incubation, remove gas-permeable seal from the second growth plate and measure end-point OD600.
Third Growth Plate
Third Growth Plate
Transfer 50 μL from each well of the second growth plate to corresponding wells of the third growth plate.
Apply gas-permeable seal to the third growth plate.
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.
Measure OD600, OD700, and fluorescence (569 nm excitation, 593 nm emission for mScarlet-I3 fluorescent protein) every 5 minutes throughout the incubation.
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).
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.
Plate Reader4 steps
For measurements with a plate reader
Plate Reader Fluorescent Measurement
Plate Reader Fluorescent Measurement
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.
In a new 96-well plate, calibrate the OD600 and fluorescence measurements using silica microspheres (950µm) and a fluorescent dye (Sulforhodamine 101) following a fluorescent calibration protocol: The reference for performing this measurement
Note: For additional instruction see References Beal 2018, Beal 2020, and Beal 2022.
Data Analysis
Data Analysis
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.
Calculate the quantitative Function by subtracting the mean fluorescent signal of the non-fluorescent control from the measured variants.
Protocol references
Tack, D. S., Tonner, P. D., Pressman, A., Olson, N. D., Levy, S. F., Romantseva, E. F., Alperovich, N., Vasilyeva, O., & Ross, D. (2021). The genotype‐phenotype landscape of an allosteric protein. Molecular Systems Biology, 17(12). https://doi.org/10.15252/msb.202110847
Beal J, Haddock-Angelli T, Baldwin G, Gershater M, Dwijayanti A, Storch M, et al. (2018) Quantification of bacterial fluorescence using independent calibrants. PLoS ONE 13(6): e0199432. https://doi.org/10.1371/journal.pone.0199432
Beal, J., Farny, N.G., Haddock-Angelli, T. et al. Robust estimation of bacterial cell count from optical density. Commun Biol 3, 512 (2020). https://doi.org/10.1038/s42003-020-01127-5
Jacob Beal, Cheryl A Telmer, Alejandro Vignoni, Yadira Boada, Geoff S Baldwin, Liam Hallett, Taeyang Lee, Vinoo Selvarajah, Sonja Billerbeck, Bradley Brown, Guo-nan Cai, Liang Cai, Edward Eisenstein, Daisuke Kiga, David Ross, Nina Alperovich, Noah Sprent, Jaclyn Thompson, Eric M Young, Drew Endy, Traci Haddock-Angelli, Multicolor plate reader fluorescence calibration, Synthetic Biology, Volume 7, Issue 1, 2022, ysac010, https://doi.org/10.1093/synbio/ysac010