Team:DTU-Denmark

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                <h2><b>Problem</b></h2>How can we quantify promoter activity?</br>
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                <p>When characterising promoters today it is done by proxy of protein. This can result in unwanted variations caused by translation and folding efficiency or excess cellular stress. Another important issue is the lack of a standard for measurements of fluorescence as most characterisations are done using fluorescent proteins such as GFP. As a result, most characterisations are done in relative units making it complicated or impossible to compare measurements between labs, strains and growth conditions. <a href="/Team:DTU-Denmark/Overview/Background"> <b>READ MORE</b> </a> </p>
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                <h2><b>Solution</b></h2>Spinach RNA allows us to measure RNA concentration using fluorometry</br>
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                <p>The Spinach RNA is an aptamer, which can bind to DFHBI to create a fluorophore that resembles GFP in spectral properties. This provides a way to easily quantify RNA concentration and allows promoter activities to be <b><a href="/Team:DTU-Denmark/Achievements/Calculator">calculated</a></b> in units Polymerases Per Second. The possibility of measuring promoter activity in absolute terms will enable researchers to share and compare results obtained in different labs, and better characterise the function of promoters. <a href="/Team:DTU-Denmark/Overview/Strategy"> <b>READ MORE</b> </a></p>
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                <h2><b>Results</b></h2>We have developed a method that can be used to measure absolute promoter activity</br>
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                <p>Using RNA transcribed <i>in vitro</i> we created a standard series which can be used to determine the concentration of Spinach given a fluorescence intensity. We also designed an experiment to determine the degradation rate of the Spinach RNA <i>in vivo</i>. Combining these two experiments with the growth rate and fluorescence of a Spinach-expressing culture it is possible to determine the promoter activity using our derived formulas or our online calculator. <a href="/Team:DTU-Denmark/Achievements/Experimental_Results"> <b>READ MORE</b> </a></p>
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<h1>Our Project</h1><br>
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<h3>Try Our PoPS Calculator!</h3>
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<p>Have you measured promoter activity but is data analysis a drag? Use our PoPS calculator to calculate promoter activity based on Spinach measurements!</p>
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<maincontentSynthetic biology is the future approach for creating biological systems with novel functions. Doing this requires a large toolbox of genetic parts, having precisely described functions.</maincontent <br>
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At the DTU iGEM team 2014, we have found that characterization of one of the most central tools is missing - genetic promoters are largely uncharacterised. Because of their importance in all synthetic biology systems, it is vital that promoter strength can be characterized in detail. Today, promoters are often used without prior characterization, sometimes based on a qualitative and relative assessment of their activity. In cases where promoters are characterized, including the iGEM Registry of Parts, a relative characterization has been used. However, relative characterizations have several major drawbacks, including lack of generalization.<br>
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During our project we seek to develop a convenient, fast and reliable method for measuring absolute promoter activities using the Spinach technology. This method relies on fluorescently tagging RNA molecules called “Spinach”. By means of the Spinach concept, we will measure the concentration of the RNA product from different promoters and use this to calculate the transcription rate of the promoters. Using standard references in our measurements will even allow us to calculate transcription rates in units of Polymerases Per Second (PoPS), which is regarded as the optimal promoter activity measure.<br>
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Latest revision as of 08:32, 7 February 2015

Technical University of Denmark

Problem

How can we quantify promoter activity?

When characterising promoters today it is done by proxy of protein. This can result in unwanted variations caused by translation and folding efficiency or excess cellular stress. Another important issue is the lack of a standard for measurements of fluorescence as most characterisations are done using fluorescent proteins such as GFP. As a result, most characterisations are done in relative units making it complicated or impossible to compare measurements between labs, strains and growth conditions. READ MORE

Solution

Spinach RNA allows us to measure RNA concentration using fluorometry

The Spinach RNA is an aptamer, which can bind to DFHBI to create a fluorophore that resembles GFP in spectral properties. This provides a way to easily quantify RNA concentration and allows promoter activities to be calculated in units Polymerases Per Second. The possibility of measuring promoter activity in absolute terms will enable researchers to share and compare results obtained in different labs, and better characterise the function of promoters. READ MORE

Results

We have developed a method that can be used to measure absolute promoter activity

Using RNA transcribed in vitro we created a standard series which can be used to determine the concentration of Spinach given a fluorescence intensity. We also designed an experiment to determine the degradation rate of the Spinach RNA in vivo. Combining these two experiments with the growth rate and fluorescence of a Spinach-expressing culture it is possible to determine the promoter activity using our derived formulas or our online calculator. READ MORE



Try Our PoPS Calculator!

Have you measured promoter activity but is data analysis a drag? Use our PoPS calculator to calculate promoter activity based on Spinach measurements!



Technical University of Denmark
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark
+45 45 25 25 25
dtuigem14@gmail.com .

Retrieved from "http://2014.igem.org/Team:DTU-Denmark"