Bacterial FACS

Procedure

Culture and induction

  1. Grow 3 mL of LB cultures of library (or positive control).

  2. Dilute overnight cultures 100-fold in fresh 3-mL LB culture.

  3. Grow for 2 hours at 37 degrees or until it reaches an optical density of 0.6.

  4. Induce for 3 hours.

Sorting

  1. Perform two successive 400x dilutions into HHBSS and screen by FACS.

  2. Sort cells into 500 μL of LB or SOB without antibiotic.

  3. Allow cells to recover at 37 Celsius with shaking for 30 minutes.

  4. Plate on LB/Agar with the appropriate antibiotic.

Notes and Cautions

  • Induction can be carried out at 25 or 37 Celsius. Inducing for 3 hours is advantageous because it also selects for quickly maturing fluorescent proteins, but can be carried out overnight as well.

  • Flow-cytometer should be configured to trigger off of side-scatter with 488-excitation, and the threshold of triggering should be decreased to just above the scatter generated by an empty droplet.

  • Forward scatter, side-scatter, and fluorescence channels should all be in ‘log’ mode.

  • It is often nice to set the sort-logic to include a side-scatter/forward-scatter gate and a red-fluorescence gate, while excluding cells that appear in the autofluorescence and green-fluorescence region.

  • Typical PMT voltages including 400 and 520 volts, for GFP and RFP, respectively.

  • A “sort single” mode is most typical, but an “enrich” mode can also be used.

  • In the literature, it has been reported that fluorescence intensity can be scaled by the scattering amplitude to account for cell size and morphology.

  • It is pretty common to only have 30-50% efficiency for sorting for bacteria on a FACS.