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Team:DTU-Denmark/Overview/Strategy
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At the Giant Jamboree, we received a gold medal and an Interlab Study Award. We also won the Overgrad Best Parts Collection.
Aims |
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Reasoning |
We argued that the activity of a constitutive promoter is determined by the concentration of free RNA polymerases in the cell and the binding affinity of the polymerase to the promoter. We further argued that the binding affinity between the promoter and polymerase is determined by promoter sequence alone, and that the number of free polymerases in the cell is strongly correlated to cell growth rate. Because of this we hypothesised that it is possible to derive a single characteristic for a constitutive promoter, which can be used to calculate promoter activity given a particular growth rate. | Reporter |
To measure promoter activity we needed to choose a reporter. Instead of using GFP or a similar fluorescent protein we chose to use an RNA reporter known as Spinach. Spinach is a non-coding RNA aptamer that fluoresces only after binding to a specific ligand. By using Spinach instead of a protein reporter we could eliminate the effects of different translational efficiencies and measure RNA concentrations directly. Measuring the RNA concentration and the degradation rate, would allow us to calculate the rate of formation of RNA, i.e. the transcription activity. To be able to correlate the measured fluorescence to actual RNA concentrations we needed a standard series. To do this we mixed excess Spinach with known concentrations of ligand, and argued that the fluorescence values measured could be directly translated to the values measured from excess ligand and limiting Spinach as found in vivo. |
Technical University of Denmark
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark
+45 45 25 25 25
dtuigem14@gmail.com .
+45 45 25 25 25
dtuigem14@gmail.com . 
