Part:BBa_K206000:Characterization

Name: pBAD strong Input: [http://openwetware.org/wiki/Arabinose L-arabinose] Output: PoPS All characterization of BBa_K206000 was done using BW27783 cells carrying the construct BBa_K206002. Transfer Function We attempted to find the transfer function relating input concentration of arabinose to PoPS by measuring RFP production in response to varying arabinose concentration. Expression Rate We measured the development of RFP over time, in response to intermediate concentrations of arabinose. Specificity We investigated the specificity of BBa_K206000 for its ligand, arabinose, by testing it against several other aldopentoses of similar structure. General Characterization Protocols 1. Inoculate 5mL LB-Chlor medium in a sterile test tube with a colony from a streak plate of pSB1C3-K206000-transformed BW27783 cells. 2. Grow overnight at 37C, shaking at 210rpm. 3. Measure OD600 and dilute to OD600=0.25A in 15mL of LB-Chlor with added arabinose (or other sugars). 4. Grow at 37C, shaking at 210rpm. 5. At appropriate timepoints (~24 hours for Transfer Function and Specificity experiments), remove samples for measuring OD600 (1/10 dilution) and fluorescence (fluorescence samples were frozen at -80C as cell pellets and measured together). Sugars were made up to 10% w/v or 500uM stock solutions in water and filter sterilized with a 0.45um filter. RFP fluorescence was measured by pelleting 500uL of cells and resuspending in PBS, then collecting 50000 events on a Becton-Dickinson Influx flow cytometer. Fluorescence was calculated by finding the mean of the RFP histogram for each sample, normalized to OD600 (when possible).