TITLE: Library Bottlenecking Protocol (Basic Microbial Culture Equipment)
AUTHORS: David Ross

[DESCRIPTION]
Protocol for bottlenecking a library using a 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
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
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
5. Prepare six new 250-mL baffled flasks, each with 50 mL media. 
Number the flasks 1 - 6
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: 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: Plate and incubate all cultures
13. Plate 150 uL from each microcentrifuge tube onto the corresponding agar plate.
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.
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
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
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: 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: 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)