Team:Wageningen UR/ATP biosensor

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    <h1>ATP biosensor</h1>
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<h1>Modeling</h1>
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<h2>“When I came out of school I didn't even think that modeling was a job.”</h2>
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            <li id="li_tab2" onclick="tab('tab2')" class="active"><a>Secondary metabolites</a></li>
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            <li id="li_tab3" onclick="tab('tab3')"><a>Toolbox</a></li>
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            <li id="li_tab4" onclick="tab('tab4')"><a>Host engineering</a></li>
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        <li class="firstbgsm fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why <i>Aspergillus nigem</i>?</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="first fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why <i>Aspergillus nigem</i>?</a></li>
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        <li class="smbg fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="first fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why <i>Aspergillus nigem</i>?</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
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        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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        <li class="smbgsm current">Modeling</li>
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        <li class="smbg"><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
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        <li class="bgbg"><a href="https://2013.igem.org/Team:Wageningen_UR/Infrastructure">Infrastructure</a></li>
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        <li class="bgsm"><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li class="last"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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    <ul>
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        <li class="first fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why <i>Aspergillus nigem</i>?</a></li>
 +
        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
 +
        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
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        <li class="smbgsm current">Modeling</li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Infrastructure">Infrastructure</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li class="smbgsm"><a href="https://2013.igem.org/Team:Wageningen_UR/Engineering_morphology">Host engineering</a></li>
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        <li class="last"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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        <li class="first fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Why_Aspergillus_nigem">Why <i>Aspergillus nigem</i>?</a></li>
 +
        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Secondary_metabolites">Secondary metabolites</a></li>
 +
        <li class="fill"><a href="https://2013.igem.org/Team:Wageningen_UR/Lovastatin">Lovastatin</a></li>
 +
        <li class="smbgsm current">Modeling</li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Biosensors">Biosensors</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Infrastructure">Infrastructure</a></li>
 +
        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Chromoproteins">Chromoproteins</a></li>
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        <li><a href="https://2013.igem.org/Team:Wageningen_UR/Engineering_morphology">Host engineering</a></li>
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        <li class="last bg"><a href="https://2013.igem.org/Team:Wageningen_UR/Summary">Summary</a></li>
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    </ul>
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    <h2>ATP bio-sensor</h2>
 
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<h2>Abstract</h2>
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Economics is a vital aspect of any system, be it the world, a country, a person or even a microbe. Fungi are being used extensively by the biotechnology industry to produce useful compounds like drugs, organic acids, enzymes, fuels, etc. Thus efficient use of cellular resources is of prime importance in these industrial workhorses. Now the energy currency of any cell is Adenosine tri-phosphate (ATP) molecule and visualizing the dynamics of ATP levels in living cells has been a challenge. To this end we aim to produce a Fluorescence resonance energy transfer (FRET) based sensor for live cell ATP measurements in Aspergillus niger. With this we hope to measure ATP levels between compartments in growing Aspergillus niger during major metabolic shifts to understand the energy management processes in the cell.
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<h2>Introduction</h2>
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== Introduction ==
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Fluorescence resonance energy transfer (FRET) is a phenomenon widely exploited by bio-sensors to monitor concentrations and temporal fluctuations of metabolites and ions at cellular and sub-cellular level. FRET works by excitation of a fluorescent molecule (donor) by a light of particular wavelength, which consequently transfers this energy to an adjacent fluorescent molecule (acceptor) that in-turn emits light. This phenomenon is very sensitive to the distance between the donor and acceptor fluorophore groups. Thus, fusing fluorescent proteins with a sensing domain that undergoes big conformational changes upon binding of the sensory molecule confers the possibility for generating an assortment of custom-made genetically encoded biosensors. These are useful tools to non-invasively quantify metabolites in living cells.  
Fluorescence resonance energy transfer (FRET) is a phenomenon widely exploited by bio-sensors to monitor concentrations and temporal fluctuations of metabolites and ions at cellular and sub-cellular level. FRET works by excitation of a fluorescent molecule (donor) by a light of particular wavelength, which consequently transfers this energy to an adjacent fluorescent molecule (acceptor) that in-turn emits light. This phenomenon is very sensitive to the distance between the donor and acceptor fluorophore groups. Thus, fusing fluorescent proteins with a sensing domain that undergoes big conformational changes upon binding of the sensory molecule confers the possibility for generating an assortment of custom-made genetically encoded biosensors. These are useful tools to non-invasively quantify metabolites in living cells.  
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<p class="caption">ATP FRET sensor (Imamura <i>et al.</i> 2009) </p>
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FRET based sensors have been recently developed to quantify ATP levels in vivo in HeLa and yeast cells (1, 2). They consist of mseCFP, the ATP sensing domain and mVenus (YFP variant). The YFP/CFP emission ratio gives an estimation of the ATP concentration.
FRET based sensors have been recently developed to quantify ATP levels in vivo in HeLa and yeast cells (1, 2). They consist of mseCFP, the ATP sensing domain and mVenus (YFP variant). The YFP/CFP emission ratio gives an estimation of the ATP concentration.
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== Aim ==
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Construct a fluorescent protein based sensor that will measure ATP levels between compartments in growing Aspergillus niger during metabolic shifts.  
Construct a fluorescent protein based sensor that will measure ATP levels between compartments in growing Aspergillus niger during metabolic shifts.  
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<h2>Applications</h2>
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== Applications ==
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An ATP bio-sensor can lead to visualization of the ATP levels in live Aspergillus cells. Inquiry into whether the single cell phenotype of Aspergillus niger still retains its metabolic activity can be carried out. It would be interesting to quantify the ATP levels in different organelles and investigate where and why such ATP levels exist. Also, the differences in cellular ATP under various growth environments can be studied. The differences in ATP levels for cells at the hyphal tip and those in the inner mycelium can be validated.   
An ATP bio-sensor can lead to visualization of the ATP levels in live Aspergillus cells. Inquiry into whether the single cell phenotype of Aspergillus niger still retains its metabolic activity can be carried out. It would be interesting to quantify the ATP levels in different organelles and investigate where and why such ATP levels exist. Also, the differences in cellular ATP under various growth environments can be studied. The differences in ATP levels for cells at the hyphal tip and those in the inner mycelium can be validated.   
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== Approach ==
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Four versions of the ATP sensor were introduced into A. niger – 1. ATP sensitive and cytoplasmic localization, 2. ATP sensitive and mitochondrial localization, 3. ATP in-sensitive and cytoplasmic localization, 4. A codon optimized version, ATP sensitive and cytoplasmic localization. The ATP sensor was expressed in Escherchia coli and purified by attaching an N-terminal Histidine tag. The purified sensor was characterized in vitro and the YFP/CFP emission ratio (at 527/475 nm respectively) to different ATP concentrations was tested at room temperature in a buffer mimicking the in-vivo conditions in A. niger.  
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Introduce bio-sensor into <i>A. niger</i> directed to cytolpasm and mitochodria:
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<p><span class="ref">1.</span>Codon optimized for <i>A. niger</i> <br \>
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<span class="ref">2.</span>Not codon optimized
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Express sensor in <i>Escherichia coli</i> and purify with the help of a Histidine tag for <i>in-vitro</i> characterization
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== Research Methods ==
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The initial target was to introduce the following versions of the ATP sensor into <i>A. niger</i> – 1. ATP sensitive and cytoplasmic localization; 2. ATP sensitive and mitochondrial localization; 3. ATP in-sensitive and cytoplasmic localization; 4. Codon optimized, ATP sensitive and cytoplasmic localization; 5. Codon optimized, ATP sensitive and cytoplasmic localization.
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Also, it was aimed to express the ATP sensor in <i>Escherchia coli</i> and purify with the help of a N-terminal Histidine tag for <i>in-vitro</i> characterization of the sensor. The <i>in-vitro</i> characterization of the purified sensor involves exciting the CFP with 435 nm light and measuring the YFP/CFP emission ratio (at 527/475 nm respectively) at different ATP concentrations. The characterization of the sensor was carried out at room temperature in a buffer mimicking the <i>in-vivo</i> conditions in <i>A. niger</i>.  
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Latest revision as of 12:55, 4 October 2013

Modeling

“When I came out of school I didn't even think that modeling was a job.”

Introduction

Fluorescence resonance energy transfer (FRET) is a phenomenon widely exploited by bio-sensors to monitor concentrations and temporal fluctuations of metabolites and ions at cellular and sub-cellular level. FRET works by excitation of a fluorescent molecule (donor) by a light of particular wavelength, which consequently transfers this energy to an adjacent fluorescent molecule (acceptor) that in-turn emits light. This phenomenon is very sensitive to the distance between the donor and acceptor fluorophore groups. Thus, fusing fluorescent proteins with a sensing domain that undergoes big conformational changes upon binding of the sensory molecule confers the possibility for generating an assortment of custom-made genetically encoded biosensors. These are useful tools to non-invasively quantify metabolites in living cells.

ATP FRET sensor (Imamura et al. 2009)

FRET based sensors have been recently developed to quantify ATP levels in vivo in HeLa and yeast cells (1, 2). They consist of mseCFP, the ATP sensing domain and mVenus (YFP variant). The YFP/CFP emission ratio gives an estimation of the ATP concentration.

Aim

Construct a fluorescent protein based sensor that will measure ATP levels between compartments in growing Aspergillus niger during metabolic shifts.

Applications

An ATP bio-sensor can lead to visualization of the ATP levels in live Aspergillus cells. Inquiry into whether the single cell phenotype of Aspergillus niger still retains its metabolic activity can be carried out. It would be interesting to quantify the ATP levels in different organelles and investigate where and why such ATP levels exist. Also, the differences in cellular ATP under various growth environments can be studied. The differences in ATP levels for cells at the hyphal tip and those in the inner mycelium can be validated.

Approach

Introduce bio-sensor into A. niger directed to cytolpasm and mitochodria:

1.Codon optimized for A. niger
2.Not codon optimized

Express sensor in Escherichia coli and purify with the help of a Histidine tag for in-vitro characterization

Research Methods

The initial target was to introduce the following versions of the ATP sensor into A. niger – 1. ATP sensitive and cytoplasmic localization; 2. ATP sensitive and mitochondrial localization; 3. ATP in-sensitive and cytoplasmic localization; 4. Codon optimized, ATP sensitive and cytoplasmic localization; 5. Codon optimized, ATP sensitive and cytoplasmic localization.

Also, it was aimed to express the ATP sensor in Escherchia coli and purify with the help of a N-terminal Histidine tag for in-vitro characterization of the sensor. The in-vitro characterization of the purified sensor involves exciting the CFP with 435 nm light and measuring the YFP/CFP emission ratio (at 527/475 nm respectively) at different ATP concentrations. The characterization of the sensor was carried out at room temperature in a buffer mimicking the in-vivo conditions in A. niger.

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