Library Bottlenecking Protocols

Dilute a full 1 mL vial of the library glycerol stock into 50 mL of media in a 250 mL baffled flask.

Incubate the resulting culture at 37 C with shaking (300 rpm) for 12 - 24 hours (to reach stationary phase).

• This generates the overnight flask

Prepare one baffled flask (125 mL or 250 mL) with 49.9 mL media

• This is the dilution flask

Prepare one baffled flask (125 mL or 250 mL) with 49.9 mL media

• This is the dilution flask

One new 250-mL baffled flask with 50 mL media.

• This is the final flask

Prepare six 15 mL snap-cap culture tubes, each with 2 mL media

• Number the tubes 1 - 6

Six 15 mL snap-cap culture tubes, each with 2 mL media and number the tubes 1 - 6.

Prepare six new 250-mL baffled flasks, each with 50 mL media. 

• Number the flasks 1 - 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

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

Three agar plates (with LB + kan) and number the plates 1, 2, 3

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)

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.

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).

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.

Swirl the dilution flask to mix well.

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.

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.

Measure a media blank sample with the flow cytometer.

• Use a 150 uL acquisition volume at 100 uL/min flow rate

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)

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

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

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)

Plate 150 uL from each microcentrifuge tube onto the corresponding agar plate.

Record the “raw cell count” as the number of events within the scattering-signal gate

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)

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

The next day, count the colonies on every agar plate to get colony forming unit (CFU) estimates for the starting point for each culture.

Choose the culture tube with closest to 20,000 cells per 100 uL.

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.

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.

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.

Add the input volume of the chosen culture to the final 250-mL baffled flask with 50 mL media.

Mix the final flask by swirling several times.

Pipette 1 mL from the final flask to microcentrifuge tube 1 (the empty one).

Mix microcentrifuge tube 1

Pipette 100 uL from microcentrifuge tube 1 to microcentrifuge tube 2 and mix.

Mix microcentrifuge tube 2

Pipette 100 uL from microcentrifuge tube 2 to microcentrifuge tube 3

Mix microcentrifuge tube 3

Plate 150 uL from each microcentrifuge tube onto the corresponding agar plate.

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).

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.

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.