Team:DTU-Denmark/Overview/Strategy

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Experimental Design

The polymerase is in the process of transcription of the Spinach DNA sequence surrounded by the tRNA scaffold for increased stability. The black arrow indicates the promoter of which the activity is about to be determined.
Internal interactions within each Spinach molecule take place and eventually the correctly folded structure occur thus allowing the later formation of a fluorophore complex. This process is an equilibrium where about 50 to 60 percent of Spinach is in the proper folded state.
Internal interactions within each Spinach molecule take place and eventually the correctly folded structure occur thus allowing the later formation of a fluorophore complex. This process is an equilibrium where about 50 to 60 percent of Spinach is in the proper folded state.
The correctly folded Spinach molecule can interact with the yellow compound DFHBI-1T to form a fluorophore complex with an excitation peak of 482nm and an emission peak at 505nm. The quantified fluorescent signal can then be converted into the absolute activity of the promoter by the means of a standard curve, copy number, stability, cell density and growth rate.
The correctly folded Spinach molecule can interact with the yellow compound DFHBI-1T to form a fluorophore complex with an excitation peak of 482nm and an emission peak at 505nm. The quantified fluorescent signal can then be converted into the absolute activity of the promoter by the means of a standard curve, copy number, stability, cell density and growth rate.

Aims

The aim of our project is twofold: Firstly, we wanted to develop a method to easily measure the activity of a given promoter in meaningful units, such as polymerases per second (PoPS). Being able to conveniently measure promoter activity in such an absolute unit, would make it easier to compare results across different labs and experiments.
Secondly, we wanted to use our absolute activity measurements to characterise promoters in the Standard Registry of Parts.

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.