Team:DTU-Denmark/Overview/Strategy

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Experimental Design

Choice of Technology

The main objective of our experimental project has been to develop a new method for quantifying promoter activities. We chose to do this by measuring the RNA concentration at steady state growth and by calculating the RNA formation rate. Thus, much of our work has concerned proof-of-concept of this new measuring method.

We decided to work with a new and promising technology: The Spinach aptamer. This is a RNA sequence that upon folding are able to bind a fluorophore in a unique complex. The Spinach RNA sequence used in our project is a derivative of a Spinach aptamer developed at Cornell University by Jaffrey et al., and the technology has been used by previous iGEM teams, the 2012 UT Austin team and 2012 Carnegie Mellon team, but with different purposes. The Spinach RNA can bind and activate the fluorophore DFHBI. Neither the unbound fluorophore nor the Spinach RNA fluoresces significantly on their own, but the complex of the two is highly fluorescent emitting a green fluorescence (505 nm).

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.

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.

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.

In our project we have chosen to work with an improved version of Spinach, the so-called Spinach2.. This aptamer has increased folding efficiency compared to the original Spinach. Furthermore we use a modified version of the fluorescent ligand, the DFHBI-1T. 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..

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 2), 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)

Figure 2: The Spinach2 sequences used in our project. From left, 1st Modified Spinach2 sequence, Original Spinach2 sequence and 2nd Modified Spinach2 Sequence.

The original Spinach2 is shown in the center of Figure 2. The red area indicates the location of the SpeI restriction site in the DNA sequence. The goal of the mutations was to remove the SpeI site while conserving the structure, folding efficiency and DFHBI-binding of the resulting RNA. The first and most promising suggestion was to swap two bases that are predicted to bind. The second strategy was to change one base into another chemically similar base, i.e. changing a uracil (U) predicted not to bind any other bases, to cytosine (C), another pyrimidine base.

RNA is usually quite unstable in vivo, which directly influences the concentrations we would be able to measure. Therefore we decided to stabilise the Spinach RNA by flanking it with a tRNA scaffold, a method that has also been employed by the inventors of Spinach.

Measuring Anderson Library Promoters

Previous work with Spinach and Spinach2 in E. coli has been done using only very strong promoters (e.g. T7 promoters and rRNA promoters) to express the aptamer. We wanted to develop a method that can also be used to measure the activity of promoters of more modest strength. We decided to use promoters from the Anderson library as representatives of E. coli housekeeping promoters of varying strength.

Using An In Vitro Standard

Since there is no well-established standard unit for measuring fluorescence, we decided to make an in vitro Spinach standard series that can be used to correlate fluorescence to RNA concentration, by adding known concentrations of DFHBI-1T to excess Spinach RNA. The advantage of using such a standard compared to a universally agreed upon, but arbitrarily chosen fluorescence unit, is that it allows calculation of RNA concentration (and hence RNA formation rate) in chemically meaningful units, that can be readily compared with concentrations obtained from other assays.