Minnesota/22 October 2008

Characterization of an existing Biobrick part: Characterization of araC promoter (R0080)

Construction of the composite part: Bba_R0080 was obtained from source plate 1001, well number 11A and transformed into Top10 cells. The plasmid was digested with EcoRI and SpeI restriction enzymes and cleaned by Ethanol precipitation. BBa_E0240 (GFP) was obtained from source plate 1001, well number 4B and transformed into Top 10 cells. Plasmid was digested dwith XbaI and PstI. The part was cleaned by gel elution. pSB3K3 plasmid was obtained from source plate 1014, well number 1F and transformed into db.1 cells. Backbone plasmid was amplified by PCR using specific primers. Amplified plasmid was digested with EcoRI and PstI and purified by gel elution. All the 3 parts above were ligated by 3 way ligation, clones obtained were tested and confirmed by PCR and restriction digestion.

Activity assay of the araC promoter: 2 clones were grown over night in minimal media (MOPs Media) with 0.4% glycerol. Next day 1% inoculum was used to inoculate 10 ml media. Cells were allowed to grow for 5 hours (OD600~ 0.4). This culture was aliquoted in a 96 well plate and 0, 5 and 10 g/ml arabinose (inducer) was added to the medium. The measurement for both the clones and for the three inducer amounts was done in triplicates. OD600 and fluorescence were measured for 3.5 hours after every 30 minutes. Fluorescence was normalized w.r.t OD600 and plotted against time (Figure.3). Promoter was found to be moderately active and no leaky expression was observed.