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.

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

Technical University of Denmark
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark

+45 45 25 25 25

dtuigem14@gmail.com .

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 <maincontent>
+<h2>Choice of Technology</h2>
+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 <a href="https://2014.igem.org/Team:DTU-Denmark/Achievements/Calculator”>calculating the RNA formation rate</a>.
+We chose to work with a new and promising technology: The Spinach aptamer (link:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314379). The Spinach RNA can bind and activate the fluorophore DFHBI (link:http://www.lucernatechnologies.com/DFHBI-1T-5mg-p29.html). 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).
 <br><br>
 <h2>Aims</h2>