Part:BBa_R0011:Experience/iGEM10 Washington

Washington 2010 R0011 in different plasmids

Overview

As part of the expression cassette BBa_K314103 the R0011 promoter was included with a Lac I generator BBa_K314111. This expression cassette was place in 4 different plasmid backbones pSB1C3, pSB1A3, pSB3K3, and pSB4A5 giving a range of copy number to test expression and repression levels in. BBa_E0040 (GFP) was placed in the expression portion of the cassette. Data is expressed below as relative fluorescence at 485(ex) 525 (em) nm over optical density at 600nm

Outline of procedures

The plasmid was place into a DH5a cell line and held at -80 in a 25% glycerol solution. The day before the assay a scraping of the glycerol stock was used to inoculate an overnight of 1ml TB with plasmid specific antibiotic. The overnight was grown at 37C on a shaker. The next day 20 ml of the overnight was added to 1ml of TB with plasmid specific antibiotic. The new inoculation was held on a shaker at Rm temperature for one hour then 50ml of 10mM IPTG was added to activate R0011 expression. The induced and un-induced samples were held at Rm temperature for 18hrs then spun down at 4000rpm for 20 minutes to pellet the cells. Supernatant was poured off and the cells were suspended in 1ml 7.5pH PBS. The cells were spun down again at 4000rpm for 20 minutes, the supernatant was removed and the cells were suspended in 1ml 7.5pH PBS. 100ul of the broth was used for OD600 reading and 100ul was used for RFU at 525nm. Cells not containing GFP and PBS without cells were used as blanks.