Difference between revisions of "Team:William and Mary/Protocols"

All time course measurements were carried out in the E. coli strain NEB 10-Beta (ΔAra-Leu) in M9 media with .1% casamino acids, .4% glucose (or glycerol when arabinose was used), and 500mM L-leucine, and were measured using a BioRad S3e Cell Sorter, with a minimum of 10,000 cells measured from each of three biological replicates (colonies) collected on the same day. Care was made to maintain cells in midlog growth phase.

The procedure for FACS time course experiments was as follows: Cells were inoculated from LB agar plates in the morning into fresh M9 containing appropriate selective antibiotics. They were then grown for between 7-10 hours in a 37C shaking incubator at 250 rpm. Then, OD600 was measured using a spectrophotometer, and cells were diluted to an OD600 of .01 into fresh inducer containing media. Time points were collected directly onto ice, and the time out of the shaking incubator for cells was kept to less than 2 minutes. Iced time points were then immediatly FACSed, and controls were performed to ensure cells were not changing fluorescence over time. Cells were diluted every 100 minutes into fresh pre-warmed M9 to maintain midlog growth.

Flow cytometry data was aquired on the FL1 (sfGFP/MEFL) or FL3 (mScarlet-I/RFP/MECY) channel, with samples being kept at 4C throughout. Voltages and gatings were standardized from early on, and were always set to the same settings (FSC/SSC: 500, FL1 gain: 600, FL3 gain: 675). At least 10,000 (typically 20,000) events/cells collected after gating were collected from each biological replicate, and at least 20,000 events were collected each day from absolute fluorescence calibration beads (SpheroTech Rainbow Calibration Particles RCP-30-5A, bead lots AH01, AB01) to allow for setting independent conversion to absolute units. FlowCal was used for downstream analysis and conversions to absolute fluorescence units. The two absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and Molecules of Equivalent CY5 (MECY) for mScarlet-I.