TITLE: Library Bottlenecking Protocols
AUTHORS: David Ross

[DESCRIPTION]
Protocol for bottlenecking a variant library for downstream use in the Pooled, Growth-Based Assay pipeline using either a positive-displacement flow cytometer or basic microbial culture equipment. 

[STEPS]
SECTION: Create the first overnight culture
1. Dilute a full 1 mL vial of the library glycerol stock into 50 mL of media in a 250 mL baffled flask.
SECTION: Prepare all flasks and tubes
4. One new 250-mL baffled flask with 50 mL media. 
This is the final flask
SECTION: Prepare all flasks and tubes
5. Six 15 mL snap-cap culture tubes, each with 2 mL media and number the tubes 1 - 6.
SECTION: Prepare all flasks and tubes
6. Three 1.5 mL microcentrifuge tubes and number the tubes 1, 2, 3
Leave tube no. 1 empty
Add 900 uL media to each of the other tubes
SECTION: Prepare all flasks and tubes
7. Three agar plates (with LB + kan) and number the plates 1, 2, 3
SECTION: Prepare all flasks and tubes
3. Prepare one baffled flask (125 mL or 250 mL) with 49.9 mL media
This is the dilution flask
SECTION: Create the first overnight culture
2. Incubate the resulting culture at 37 C with shaking (300 rpm) for 12 - 24 hours (to reach stationary phase).
This generates the overnight flask
SECTION: Calibrate the flow cytometer
8. Run the startup and performance test protocols for the flow cytometer.
Also, run a blank sample, 1 mL of focusing fluid to make sure the system is well warmed up and running properly. 
SECTION: Make culture dilutions
9. Mix the 50 mL overnight culture well, then pipette 0.1 mL from the overnight flask culture into the dilution flask (with 49.9 mL media) and swirl the flask to mix well.
SECTION: Make culture dilutions
10. Make a 3-fold serial dilution series from the dilution flask:
Pipette 1 mL from extra flask to culture tube no. 1
Mix culture tube no. 1 well
Pipette 1 mL from culture tube no. 1 to culture tube no. 2
Mix culture tube no. 2 well
Pipette 1 mL from culture tube no. 2 to culture tube no. 3
Mix culture tube no. 3 well
Continue this for the remaining tubes 4-6.
SECTION: Measure samples using the flow cytometer
11. Measure a media blank sample with the flow cytometer.
Use a 150 uL acquisition volume at 100 uL/min flow rate
SECTION: Measure samples using the flow cytometer
12. Measure cultures tubes 1-6 with the flow cytometer:
Mix each tube well by vortexing before measurement.
Measure tubes in reverse order (i.e., start with tube 6).
Use a 150 uL acquisition volume at 100 uL/min flow rate
SECTION: Analyze the data
13. For the media blank and each culture tube, analyze the cytometry data to calculate the apparent number of cells per 100 uL.
Ignore the first 30 s of data for each sample
Adjust the flow cytometry gates based on the scattering signals for the culture tube samples, and use the same gate for all samples (including the media blank)
SECTION: Analyze the data
14. Record the “raw cell count” as the number of events within the scattering-signal gate
SECTION: Analyze the data
15. For the culture tube samples, subtract the number of events recorded with the media blank sample to calculate the number of cells per 100 uL for each sample 
SECTION: Analyze the data
16. Choose the culture tube with closest to 20,000 cells per 100 uL. 
SECTION: Analyze the data
17. Calculate the volume of the chosen culture required for 200,000 cells.
This is the input volume
200,000 is used here instead of 100,000 because some variants will have multiple copies in the culture. So, the counted number of cells will be an overestimate of the resulting bottleneck diversity.
SECTION: Dilute and plate cultures
18. Add the input volume of the chosen culture to the final 250-mL baffled flask with 50 mL media.
SECTION: Dilute and plate cultures
20. Pipette 1 mL from the final flask to microcentrifuge tube 1 (the empty one).
SECTION: Dilute and plate cultures
22. Pipette 100 uL from microcentrifuge tube 1 to microcentrifuge tube 2 and mix.
SECTION: Dilute and plate cultures
23. Mix microcentrifuge tube 2
SECTION: Dilute and plate cultures
24. Pipette 100 uL from microcentrifuge tube 2 to microcentrifuge tube 3
SECTION: Dilute and plate cultures
25. Mix microcentrifuge tube 3
SECTION: Dilute and plate cultures
26. Plate 150 uL from each microcentrifuge tube onto the corresponding agar plate.
SECTION: Dilute and plate cultures
27. Incubate the bottlenecked culture in the final 250 mL baffled flask at 37 C with shaking (300 rpm) for 16 - 24 hours (to reach stationary phase).
SECTION: Count cultures to verify flow cytometry data
28. The following day, count the number of colonies on each plate and use the results to estimate the number of cells that were initially diluted into the bottlenecked culture.
SECTION: Make stocks
29. Make several 1 mL glycerol stocks (0.5 mL cells + 0.5 mL 40% glycerol) from the bottlenecked culture, label appropriately, and store at -80C until use.
SECTION: Dilute and plate cultures
21. Mix microcentrifuge tube 1
SECTION: Dilute and plate cultures
19. Mix the final flask by swirling several times.
SECTION: Prepare all flasks and tubes
3. Prepare one baffled flask (125 mL or 250 mL) with 49.9 mL media
This is the dilution flask
SECTION: Prepare all flasks and tubes
4. Prepare six 15 mL snap-cap culture tubes, each with 2 mL media
Number the tubes 1 - 6
SECTION: Prepare all flasks and tubes
5. Prepare six new 250-mL baffled flasks, each with 50 mL media. 
Number the flasks 1 - 6
SECTION: Prepare all flasks and tubes
6. Prepare 18 1.5 mL microcentrifuge tubes
Number the tubes 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, …, 6.1, 6.2, 6.3
Leave tubes 1.1, 2.1, 3.1, …, 6.1 empty
Add 900 uL media to each of the other tubes
SECTION: Prepare all flasks and tubes
7. Prepare 18 agar plates (with LB + kan)
Number the plates to match the microcentrifuge tubes (1.1, 1.2, 1.3, 2.1, 2.2, 2.3, …, 6.1, 6.2, 6.3)
SECTION: Make culture dilutions
8. Mix the 50 mL overnight flask well, then pipette 0.1 mL of the extra culture into the dilution flask (with 49.9 mL media).

SECTION: Make culture dilutions
9. Swirl the dilution flask to mix well.
SECTION: Make culture dilutions
10. Make a serial dilution series from the dilution flask into culture tubes:
Pipette 1 mL from dilution flask to culture tube no. 1
Mix culture tube no. 1 well
Pipette 1 mL from culture tube no. 1 to culture tube no. 2
Mix culture tube no. 2 well
Pipette 1 mL from culture tube no. 2 to culture tube no. 3
Mix culture tube no. 3 well
Continue this for the remaining tubes 4-6.
SECTION: Make culture dilutions
11. Transfer 1 mL of each culture from tubes 1-6 to flasks 1-6 and microcentrifuge tubes 1-6 :
Mix well
Pipette 1 mL from each culture tube to corresponding baffled flask 
Mix baffled flask well
Pipette 1 mL from flask to corresponding empty microcentrifuge tube (X.1)
SECTION: Make culture dilutions
12. For each set of microcentrifuge tubes:
Mix microcentrifuge tube X.1
Pipette 100 uL from tube X.1 to tube X.2 
Mix microcentrifuge tube X.2
Pipette 100 uL from tube X.2 to tube X.2 
Mix microcentrifuge tube X.2
Pipette 100 uL from tube X.2 to tube X.3 
Mix microcentrifuge tube X.3
SECTION: Plate and incubate all cultures
13. Plate 150 uL from each microcentrifuge tube onto the corresponding agar plate.
SECTION: Plate and incubate all cultures
14. Incubate all agar plates at 37 C for 16-24 hours or until colonies are visible and incubate all flasks at 37 C with shaking (300 rpm) for 16-24 hours (to stationary phase)
SECTION: Count colonies and estimate colony forming units
15. The next day, count the colonies on every agar plate to get colony forming unit (CFU) estimates for the starting point for each culture.
SECTION: Count colonies and estimate colony forming units
16. Choose the culture with an estimated starting CFU count closest to the target diversity (100,000).
The true CFU count should be an overestimate of the library diversity because some variants will have multiple copies in the culture. But in our experience, the diversity resulting from this method is just as likely to be higher or lower than the estimated CFU count.
SECTION: Make glycerol stocks
17. Make several 1 mL glycerol stocks (0.5 mL cells + 0.5 mL 40% glycerol) from the chosen bottlenecked culture, label appropriately, and store at -80 C until use.
Also, consider making glycerol stocks from some of the other bottlenecked cultures in case you decide to run a similar library with higher or lower diversity after the initial pilot-scale pooled assay.