Team:DTU-Denmark/Overview/Strategy

From 2014.igem.org

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<pageheader>Policy and Practices</pageheader>
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<th colspan="2" scope="col"><h2>HUMAN PRACTICE - INSPIRE YOUNGER STUDENTS</h2></th>
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<th colspan="2" scope="col"><h2>Aims</h2></th>
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<th scope="col">We are a team of very different personalities and backgrounds. But one of the main parameters that makes this dynamic team such a strong unit is a great mutual interest in synthetic biology. A curiosity and an engagement in the field that made us participate in iGEM bring us together. We have a natural urge to spread this interest and enthusiasm and inspire younger student to follow the same path. In order to do so we arranged different initiatives for outreach. <br><br></th><tr><td></td></tr>
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<th scope="col">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.<br>
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Secondly, we wanted to use our absolute activity measurements to characterise promoters in the Standard Registry of Parts.<br><br></th><tr><td></td></tr>
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<th colspan="2" scope="col"><h2>Communication strategy </h2></th>
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<th colspan="2" scope="col"><h2>Reasoning</h2></th>
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<th scope="col">We believe that inspiration is an essential step towards getting younger student interested and involved in the world of synthetic biology. We emphasise the importance of inspiration before education. Our strategy has therefore been to draw students’ attention with great iGEM and synthetic biology examples and the huge future potential for synthetic biology. We believe that younger students are more disposed to search for information on the topic on a later stage as well as absorb information they are disposed to if the inspiration succeed. We take it up on a higher level of abstraction instead of focusing on the hard core theory behind synthetic biology.<br><br></th><tr><td></td></tr>
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<th scope="col">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.<br><br></th><tr><td></td></tr>
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<th colspan="2" scope="col"><h2>Reporter</h2></th>
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<th scope="col">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.<br>
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Measuring the RNA concentration and the degradation rate, would allow us to calculate the rate of formation of RNA, i.e. the transcription activity.<br>
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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.<br><br></th><tr><td></td></tr>
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Revision as of 08:36, 12 August 2014

Technical University of Denmark

Experimental Design


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.


Experimental Design


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.

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