Team:Virginia Commonwealth/Methods



Protocols for the characterization of transcription initiation were based on the methods of Kelly et al., 2009 (1), but added rtPCR analysis to determine transcript copy number of the reporter mRNA. We propose this methodology as a minimal measurement standard for promoters and other transcriptional parts.

Cell Growth

 * 1) LB broth with an appropriate selective antibiotic was used in all cell growth experiments.
 * 2) From streaked plates, three colonies were picked for each condition tested, and cells were cultured overnight (~12hrs) in liquid media.
 * 3) All samples were diluted 100x in fresh LB (time = 0).
 * 4) At time = 3hr, samples were collected for analysis of optical density, RNA purification, and RFP fluorescence.

mRNA

 * 1) The QIAGEN RNAProtect and RNeasy protocols were followed for total RNA extraction, using 6 x 10^8 cells from each sample.  Purified RNA was eluted with 50uL DEPC-treated water, and absorbance at 260 and 280nm was used to determine RNA concentration and purity.
 * 2) rtPCR analysis was performed by the Nucleic Acids Research Facility at VCU. NARF also designed and ordered primers and probes for their analysis.
 * 3) The Anderson promoter BBa_J23100 was used as the reference for rtPCR analysis, and results were normalized to total RNA recovered for each sample.

Protein

 * 1) To prepare cells for flow cytometer analysis for RFP fluorescence concentration, 0.5 mL of sample was pelleted by centrifugation and resuspended in 1.0 mL phosphate buffered saline (PBS), for a 2x dilution.
 * 2) Cells were analyzed on a BD FACScalibur flow cytometer kindly provided by Dr. John Ryan, stopping at 100,000 events or 45 seconds. Voltage settings were as follows:
 * 3) * FSC: E00
 * 4) * SSC: 350
 * 5) * FL-1: 474
 * 6) * FL-2: 550
 * 7) * FL-3: 750
 * 8) The geometric mean of each sample was recorded and compared to that of the reference Anderson promoter  BBa_J23100.
 * 9) E. coli NEB10beta cells growing on LB were used as a negative control.