Sphero rainbow calibration particles

Cells
were analyzed and
sorted with a BD FACSAria IIu flow cytometer (Becton Dickinson (BD),
Franklin Lakes, NJ). A blue solid-state laser (488 nm excitation)
and a 530/30 nm filter was used to measure eGFP. FCS files were analyzed
using Flowing Software v2.5.1 (Cell Imaging Core, Turku Centre for
Biotechnology, Turku, Finland). For flow cytometry sampling, the geometric
mean of the FITC-A fluorescence for 10 000 events was taken
as the “promoter activity”. Prior to sorting, the cytometer
was calibrated using Accudrop beads (BD) and SPHERO Rainbow calibration
particles (Spherotech, Lake Forest, IL).
On the day of sorting,
2–4 mL of library frozen stocks were thawed, centrifuged to
remove excess glycerol, and used to inoculate 25 mL of MOPS minimal
medium supplemented with 0.4% (w/v) xylose, 100 μg/mL ampicillin,
and 0.1 mM IPTG. Cells were monitored hourly until they reached a
postrecovery state (∼5 h), as indicated by 85–90% of
cells expressing GFP. Cells were then dosed with 0 or 100 μM
formaldehyde and sorted 2 h later (Figure S8). The final promoter library in the NEB5α and ΔfrmR strains was sorted into eight gates with approximately
equivalent populations, and 1 000 000 events were collected
from each gate directly into LB medium. Populations were recovered
at 37 °C overnight and miniprepped for sequencing. Plating indicated
that approximately 70% of the sorted cells survived.

The cells were prepared for flow cytometry analysis 48 h after transfection by removing the medium and incubating the cells with 200 μl StemPro^™ Accutase^™ Cell Dissociation Reagent (Gibco, cat # A11105-01) at 37°C for 5 minutes. After incubation, the plates were transferred on ice. To avoid potential cell damage the samples were prepared in successive batches so that no single sample was kept on ice for more than 1 h. The prepared samples were measured using a BD LSR Fortessa II Cell Analyzer with a combination of excitation and emission that minimizes the crosstalk between different fluorescent reporters. Cerulean and AmCyan were measured with a 445 nm excitation laser coupled to a 473/10 nm emission filter. mCherry was measured with a 561 nm excitation laser coupled to a 600 nm longpass filter and 610/20 emission filter. The Cerulean and the mCherry were measured, respectively, at PMT voltage of 330 and 310 in all the experiments except for the experiment presented in Supplementary Fig. 4a, in which mCherry was measured at PMT voltage of 325. The data presented in Supplementary Fig. 6 were obtained by measuring the AmCyan and mCherry, respectively, at PMT voltage of 350 and 315. SPHERO RainBow Calibration particles (Spherotech; Cat # RCP-30-5A, BD) were used to ensure constant device performance.