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.
- Liquid cultures
- Pick colonies from plate
- Multiple controls
- Overnight in the 37 incubator
- Liquid cultures of already grown liquid cultures
- 100 uL of grown liquid cultures into 5 mL of media + CamR
- Incubate cultures for another 4-6 hours
- Spin down cultures
- 3000 rpm for 5 minutes
- Resuspend in 1x Buffer
- 4 mL of 1x PBS
- Pipette or vortex to resuspend cells
- Plate reading
- Take 100 uL of the resuspended cells
- Load into a 96 well transparent plate
- Measure absorbance and fluorescence with GFP assay (excitation: 495 nm, emission: 509 nm)
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.