Team:DTU-Denmark/Overview/Strategy

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Experimental Design

Choice of Technology

The main objective of our experimental project was to develop a method for quantifying promoter activities. We decided to do this by measuring the RNA concentration at steady state growth and calculating the RNA formation rate We chose to work with a new and promising technology: The Spinach aptamer
The Spinach RNA can bind and activate the fluorophore DFHBI. Neither the unbound fluorophore nor Spinach RNA fluoresces significantly on their own, but the complex of the two is highly fluorescent with a green colour (505 nm). #Interactive experimental design overview:

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.

Figure 1) The transcription of the Spinach construct through folding and finally to fluorescence. The illustration is interactive and further description can be found on individual elements.

Traditionally promoter activities have been measured either by measuring the concentration of a reporter protein, e.g. a fluorescent protein such as green fluorescent protein (GFP), or by RNA quantification with quantitative PCR. The Spinach technology combines the best of both worlds:

Spinach concentration can be measured with an easy fluorescence assay using GFP filters
Promoter activity is measured directly on RNA instead of by proxy of a protein. This means that translation efficiency, a potential source of variation, can be disregarded.

In our project we have chosen to work with an improved version of Spinach, the so called Spinach2 (Link to Spinach2 article). This aptamer has increased folding efficiency compared to the original Spinach. Furthermore we use a modified version of the fluorescent ligand, the DFHBI-1T (link to Lucerna). This modified fluorophore has increased brightness when bound to Spinach2 and shows spectral properties that more closely resemble GFP, which means that higher signals can be obtained using regular GFP fluorescence filters (Link to Plug and play article).

Design of Spinach Sequence

Spinach2 contains a SpeI restriction site, which makes it incompatible with the iGEM Standard Assembly protocol. In order to be able to submit Spinach2 as a BioBrick, we decided to remove the SpeI restriction site by introducing specific point-mutations into the Spinach2 sequence (see figure XX), thereby creating two new Spinach2 versions:

We swapped an A and a U (1st Modified Spinach2, later referred to as Spinach2.1)
We exchanged a U with a C (2nd Modified Spinach2)

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.