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.

We diluted 100µL of cell culture into 400 µL of Phosphate Buffered Saline (PBS). For our induction experiments, we split and induced the cells with IPTG 6 hours after they were inoculated. Four hours after the cells were induced, we diluted the cells in order to obtain an ideal concentration of OD600 0.01. The cells were then grown for another five hours and taken out every 20 minutes to be filtered on ice and FACSed immediately. To acquire data with flow cytometry, we adjust side-scatter (SSC) and forward scatter (FSC) PMT voltages using bacteria from one of our samples until the distribution of each is centered on the scale and we adjust FITC/GFP PMT voltage until the upper edge of the “bell curve” from the fluorescent population is one order of magnitude below the upper end of the scale. We run the program enough to acquire at least 10,000 events for each biological sample. After running the samples, we acquire at least 10,000 events from a sample of calibration beads with the voltages for FSC at 339 and SSC at 292 and at the voltages applied for the sample collection on the channel(s) used. We used SpheroTech Rainbow Calibration Particles RCP-30-5A for fluorescent calibration beads because they have been calibrated for excitation and detection of the particles in most channels of any flow cytometer. Every 100 minutes, we diluted the cells 1:10 in pre-warmed culture. We used FlowCal for downstream analysis and conversion to absolute fluorescence units. The two absolute fluorescence units we used are Molecules of Equivalent Fluorescein (MEFL) for GFP and MEPTR for mScarlet.

PCR

Gel Electrophoresis

DpnI Digestion

PCR Purification

Gibson Assembly

Chemical Transformation

Inoculation

Qiagen Miniprep

Plate Reader