Difference between revisions of "Team:Bielefeld-CeBiTec/Project/PRIA"

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           <h2>Introduction</h2>
 
           <h2>Introduction</h2>
 
       </div>
 
       </div>
       <p>Our second approach to cell free detection of analytes is an approach based on the interaction of an repressor that binds to a plasmid or another DNA. Upon binding of the analyte the repressor changes its conformation and the bond to the DNA is disrupted. We call this approach Plasmid Repressor Interaction Assay. </br>
+
       <p>Our second approach to cell free detection of heavy metals and date rape drugs is based on the interaction of an repressor that binds to a plasmid or another DNA fragment. The repressor changes its conformation upon binding of the analyte  and the bond to the DNA is disrupted. The disruption of this bond will be detecetd by monitoring a loss of signal either due to the elution of fluorescent protein or due to elution of labeled DNA. We call this system <em>P</em>lasmid <em>R</em>epressor <em>I</em>nteraction <em>A</em>ssay (PRIA). </br>
The advantage over the cell extract is, that this approach does not required transcription or translation of a reporter protein and therefore the signal can be measured much faster. Furthermore, it works with just two purified components, thereby further minimizing the risk of releasing genetically modified organisms. GMOs into the wild.  
+
    The advantage over the cell extract is, that this method does not require transcription or translation of a reporter protein and therefore the signal can be measured much faster. For a similar system measurement of arsenic and cadmium contamination of water was possible within 15 to 30 minutes (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/PRIA#Siddiki2011">Siddiki <i>et al.</i> 2011</a>), thereby meeting one of the requirements often mentioned by potential users in our survey: speed.(Check out our <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/StreetScience#Speed" target="_blank">survey</a> on synthetic biology and biosensors) </br>
 +
    Furthermore, PRIA works with just two purified components, thereby minimizing the risk of releasing genetically modified organisms GMOs into the wild. Our system is not even subject to the regulations of the German "Gentechnikgesetz" (<a href="http://www.gesetze-im-internet.de/bundesrecht/gentg/gesamt.pdf" target="_blank">Genetic engineering Act</a>) This system should be robust against environmental factors, as long as they are not denaturating the protein and show increased stability and durability compared to conventional biosensors, since there are no metabolic pathways involved.(Interview with expert) It can be assumed that PRIA can still perform at high percentages of toxic substances, since neither translation nor transcription are required for the detection mechanisms.
  
 
</p>
 
</p>
 
       <div class="Subtitle">
 
       <div class="Subtitle">
 
           <h2>Aim</h2>
 
           <h2>Aim</h2>
           <p>Our aim was to develop a sensor based on the disruption of the repressor-plasmid bond. Since paper based systems are increasing in populartiy, because they are cheap and easily renewable, we wanted to establish a paper-based test strip.</p>
+
           <p>Our aim was to develop a sensor based on the disruption of the repressor-plasmid bond. Since paper based systems are increasing in populartiy because they are cheap and easily renewable, we wanted to establish a paper-based test strip, which enables swift detection of analytes by a loss of fluorescence signal.</p>
 
       </div>
 
       </div>
 
<div class="Subtitle">
 
<div class="Subtitle">
 
           <h2>Strategies</h2>
 
           <h2>Strategies</h2>
           <p>We started our experiments with a good characterized model system for repression and derepression: the lac operon and its repressor LacI. LacI immobilized on a Ni NTA column delivered the first proof of concept. There are two possible options for the transfer of this system from an impractial Ni NTA column to paper. The first one would be to immobilize the repressor on Paper, the second is to immobilize the DNA on Paper </p>
+
           <p>We started our experiments with a good characterized model system for repression and derepression: the <i>lac</i> operon and its repressor LacI. LacI immobilized on a Ni-NTA agarose column delivered the first proof of concept: Plasmid DNA containing the <i>lac</i> promoter was bound to LacI and could be detected in the elution fraction upon addition of Isopropyl-&beta;-D-thiogalactopyranosid (IPTG) to the column. The protocol applied was similar to that of standard protein-protein interaction studies. </br>
 +
There are two possible options for the transfer of this system from an impractial Ni-NTA agarose column to paper. The first one would be to immobilize the repressor protein on paper, the second is to immobilize the DNA with the operator on paper. </p>
  
 
</div>
 
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<div class="col-md-6">
 
<div class="col-md-6">
 
<h3>Immobilized Repressor</h3>
 
<h3>Immobilized Repressor</h3>
<p>Our approach was built on Fusionproteins of the repressor proteins with cellulose binding domains (CBD). By adding these proteins to paper, we hoped to immobilize them. </p>
+
<p>Our approach was built on fusion proteins of the repressor proteins with cellulose binding domains (CBD). By adding these proteins to paper, we hoped to immobilize them in a functional form. Released labeled DNA was to be detected upon analyte addition.</p>
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2015/b/b9/PRIA_immobProtein_imagesonly.gif" alt="Sorry, cannot load this file at the moment" width="350 px">
 
<img src="https://static.igem.org/mediawiki/2015/b/b9/PRIA_immobProtein_imagesonly.gif" alt="Sorry, cannot load this file at the moment" width="350 px">
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<div class="Subtitle">
 
<div class="Subtitle">
 
<h3>Immobilized DNA</h3>
 
<h3>Immobilized DNA</h3>
<p>Since the immobilization on ssDNA onto paper has been reported before (Araújo <i>et al.</i>) we aimed at using this system for the immobilization of dsDNA and combine it with an approach based on the measurement of the disruption of the binding of an GFPtagged repressor to immobilzied DNA (Siddiki <i>et al.</i>)
+
<p>The immobilization on ssDNA onto paper has been reported before (<a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/PRIA#Araújo2012">Araújo <i>et al.</i> 2012</a>) We adopted this method to enable the immobilization of dsDNA and combine it with an approach based on the measurement of the disruption of the binding of an GFP-tagged repressor protein to immobilized DNA as reported before (Siddiki <i>et al.</i>).
 
<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2015/d/d8/Bielefeld-CeBiTec_PT_PRIA_immobDNA.gif" alt="Sorry, cannot load this file at the moment" width="350 px" align="middle">
 
<img src="https://static.igem.org/mediawiki/2015/d/d8/Bielefeld-CeBiTec_PT_PRIA_immobDNA.gif" alt="Sorry, cannot load this file at the moment" width="350 px" align="middle">
<figcaption>Illustration of PRIA with immobilized DNA.</figcaption>
+
<figcaption>Illustration of PRIA with immobilized DNA and a sfGFP-tagged repressor protein bound to it. The signal measured is generated by the release of the tagged protein.</figcaption>
 
</figure></p>
 
</figure></p>
 
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           <h2>Outview</h2>
 
           <h2>Outview</h2>
 
       </div>
 
       </div>
       <p>We established the proof of concept for PRIA, and we showed that the immobilization of dsDNA on paper is feasible. We have sfGFPtagged repressor proteins, so the next step would be to bring these aspects together and optimize a protocol that allows the establishment of the protein DNA complex on paper.</p>
+
       <p>We established the proof of concept for PRIA, and we showed that the immobilization of dsDNA on paper is feasible. We have sfGFP-tagged repressor proteins, so the next step will be to bring these aspects together and optimize a protocol that allows the establishment of the protein DNA complex on paper.</p>
  
 
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<a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/PRIA"><img src="https://static.igem.org/mediawiki/2015/5/51/Bielefeld-CeBiTec_iGEM_logo.png"><p>Results</p></a>
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<a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/PRIA"><img src="https://static.igem.org/mediawiki/2015/e/eb/Bielefeld_CeBiTec_PT_PRIA_Kreis.jpg"><p>Results</p></a>
 
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<a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals"><img src="https://static.igem.org/mediawiki/2015/e/eb/Bielefeld_CeBiTec_PT_PRIA_Kreis.jpg"><p>What did we detect with these systems?</p></a>
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<a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals"><img src="https://static.igem.org/mediawiki/2015/5/51/Bielefeld-CeBiTec_iGEM_logo.png"><p>What did we detect with these systems?</p></a>
  
 
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Revision as of 09:49, 10 September 2015

iGEM Bielefeld 2015


Plasmid Repressor Interaction Assay

A cell free detection system based on two purified components

Introduction

Our second approach to cell free detection of heavy metals and date rape drugs is based on the interaction of an repressor that binds to a plasmid or another DNA fragment. The repressor changes its conformation upon binding of the analyte and the bond to the DNA is disrupted. The disruption of this bond will be detecetd by monitoring a loss of signal either due to the elution of fluorescent protein or due to elution of labeled DNA. We call this system Plasmid Repressor Interaction Assay (PRIA).
The advantage over the cell extract is, that this method does not require transcription or translation of a reporter protein and therefore the signal can be measured much faster. For a similar system measurement of arsenic and cadmium contamination of water was possible within 15 to 30 minutes (Siddiki et al. 2011), thereby meeting one of the requirements often mentioned by potential users in our survey: speed.(Check out our survey on synthetic biology and biosensors)
Furthermore, PRIA works with just two purified components, thereby minimizing the risk of releasing genetically modified organisms GMOs into the wild. Our system is not even subject to the regulations of the German "Gentechnikgesetz" (Genetic engineering Act) This system should be robust against environmental factors, as long as they are not denaturating the protein and show increased stability and durability compared to conventional biosensors, since there are no metabolic pathways involved.(Interview with expert) It can be assumed that PRIA can still perform at high percentages of toxic substances, since neither translation nor transcription are required for the detection mechanisms.

Aim

Our aim was to develop a sensor based on the disruption of the repressor-plasmid bond. Since paper based systems are increasing in populartiy because they are cheap and easily renewable, we wanted to establish a paper-based test strip, which enables swift detection of analytes by a loss of fluorescence signal.

Strategies

We started our experiments with a good characterized model system for repression and derepression: the lac operon and its repressor LacI. LacI immobilized on a Ni-NTA agarose column delivered the first proof of concept: Plasmid DNA containing the lac promoter was bound to LacI and could be detected in the elution fraction upon addition of Isopropyl-β-D-thiogalactopyranosid (IPTG) to the column. The protocol applied was similar to that of standard protein-protein interaction studies.
There are two possible options for the transfer of this system from an impractial Ni-NTA agarose column to paper. The first one would be to immobilize the repressor protein on paper, the second is to immobilize the DNA with the operator on paper.

Immobilized Repressor

Our approach was built on fusion proteins of the repressor proteins with cellulose binding domains (CBD). By adding these proteins to paper, we hoped to immobilize them in a functional form. Released labeled DNA was to be detected upon analyte addition.

Sorry, cannot load this file at the moment
Illustration of PRIA with repressor protein bound to a cellulose matrix. The signal measured is generated by the release of plasmid DNA.

Immobilized DNA

The immobilization on ssDNA onto paper has been reported before (Araújo et al. 2012) We adopted this method to enable the immobilization of dsDNA and combine it with an approach based on the measurement of the disruption of the binding of an GFP-tagged repressor protein to immobilized DNA as reported before (Siddiki et al.).

Sorry, cannot load this file at the moment
Illustration of PRIA with immobilized DNA and a sfGFP-tagged repressor protein bound to it. The signal measured is generated by the release of the tagged protein.

Outview

We established the proof of concept for PRIA, and we showed that the immobilization of dsDNA on paper is feasible. We have sfGFP-tagged repressor proteins, so the next step will be to bring these aspects together and optimize a protocol that allows the establishment of the protein DNA complex on paper.

References

Araújo, Ana Caterina; Song, Yajing; Lundeberg, Joakim; Stahl, Patrik L.; Brumer, Harry (2012): Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport. In Anal. Chem. 2012, 84, pp. 3311-3317. DOI: 10.1021/ac300025v

Lehtiö, Janne; Wernerús, Henrik; Samuelson, Patrik; Teeri, Tuula T.; Stahl, Stefan (2001): Directed immobillization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain. In FEMS Microbiol Lett. 2001, 195(2), pp. 197-204. DOI: 10.1111/j.1574-6968.2001.tb10521.x 197-204

Siddiki, Mohammad Shohel Rana; Kawakami, Yasunari; Ueda, Shunsaku; Maeda, Isamu (2011): Solid Phase Biosensors for Arsenic or Cadmium Composed of A trans Factor and cis Element Complex.In Sensors 2011, 11, pp. 10063-10073. 10.3390/s111110063