Team:Austin UTexas LASA/Contribution
Contribution
Group: Austin_UTexas_LASA 2017
Summary: We validated the promoter function by a fluorescence detection assay utilizing green fluorescent protein (GFP)-fused construct to BBa_I14034 promoter part.
This construct is produced by combining BBa_114034 promoter part and a GFP coding sequence. This plasmid contains ColE origin and CamR resistance.
Three negative control constructs containing different promoter sequences were used and compared in this assay: 1) T7 promoter (Invitrogen, USA), 2) pCP25 (BBa_K1509003) and 3) pPA3 promoter (BBa_K2458000). These three promoter do not contain GFP coding sequence.
For the fluorescence detection, we used a plate reader Infinite M200pro (TECAN Trading, Switzerland) which is equipped at the Ellington lab in UT Austin.
Because the part’s description stated that part BBa_I14034 is designed as a constitutive promoter, we did not add Isopropyl b-D-1-thiogalactopyranoside (IPTG) as a inducer for gene expression.
The plate reader measured the absorbance and fluorescence of each cell containing the BBa_I14034::GFP construct and non-GFP construct. Value of fluorescence was divided by cell density (absorbance) and the final result was shown as a graph in Figure 1 (below). The graph in figure 2 (below) shows a close-view of the fluorescence measured of the GFP driven by the control promoters.
RFU (relative fluorescence units) = value of fluorescence/value of absorbance
The result demonstrates that BBa_I14034 promoter is working to expression GFP in bacterial cells. Level of GFP expression was around 9,500 RFU. Cells containing non-GFP controls (T7::empty construct, CP25::empty construct, and PA3 empty construct) did not show significant detections.
