Part:BBa_I14034

P(Kat)

Constitutive Promoter, Medium Transcription

Usage and Biology

promoter for kanamycin resistance Sequence and Features BBa_I14034 SequenceAndFeatures

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.

Protocol

1. Liquid cultures
2. Pick colonies from plate
3. Multiple controls
4. Overnight in the 37 incubator
5. Liquid cultures of already grown liquid cultures
6. 100 uL of grown liquid cultures into 5 mL of media + CamR
7. Incubate cultures for another 4-6 hours
8. Spin down cultures
9. 3000 rpm for 5 minutes
10. Resuspend in 1x Buffer
11. 4 mL of 1x PBS
12. Pipette or vortex to resuspend cells
13. Plate reading
14. Take 100 uL of the resuspended cells
15. Load into a 96 well transparent plate
16. 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.