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

(Created page with "{{Bielefeld-CeBiTec/Header}} <html> <head> </head> <body> <div class="container"> <div class="jumbotron"> <h1>PRIA</h1> <p>Plasmid-Repressor...")
 
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           <h2>Successful detection of an Analyte <i>in vitro</i> </h2>
 
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      <p>The main achievement was the establishment of the PRIA. We demonstrated that the detection of analytes in drinking water is feasible in a biological system without transcription or translation of reporterproteins, but just by detecting the disruption of the bond between plasmid DNA and a repressorprotein.</p>
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          <h2>Successful Expression and purification of functional sfGFP-tagged repressorproteins <i>in vitro</i> </h2>
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          <p>The repressor for arsenic and the Repressor of the <i>blc</i> operon, as well as our model protein LacI were tagged with a sfGFP c-terminally. Their spectra were characterized and their functionality could be proved by EMSA. LacI-sfGFP and arsR showed a clear EMSA shift. </p>
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          <h2>Successful immobilization of DNA on paper </h2>
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      <p> Based on a method proposed by Araújo <i>et al.</i> (Araújo <i>et al.</i>, 2012) DNA was immobilized on Whatman Filter paper previously activated wth p-phenylene-diisothiocyanate. We made some adaptations in order to immobilise dsDNA instead of ssDNA.</p>
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          <h2>Successful immobilization of Protein on paper </h2>
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      <div><p> We pursued our second approach with was based on repressors fused to a cellulose binding domain (BBa_K1321340). All constructs were cloned successfully. When pipetted into
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          <h2>Successful simultaneous visualization of Protein and DNA on paper</h2>
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      <p>Since the repressorproteins we wanted to detect were tagged with sfGFP, they were detectable via fluorescence. DNA was labeled with Cy3 containing primers, therefore it was also detectable on paper. Both components were visualized with the Ettan Dige. The exposure time was optimized as was the paper that was used. </p>
 
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    <h1>References</h1>
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        <p>Araújo <i>et al.</i>, 2012, Activated Paper Surfaces for the rapid hybridization of DNA through Capillary Transport</p>
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Revision as of 21:34, 20 August 2015

iGEM Bielefeld 2015


PRIA

Plasmid-Repressor-Interaction Assay

Successful detection of an Analyte in vitro

The main achievement was the establishment of the PRIA. We demonstrated that the detection of analytes in drinking water is feasible in a biological system without transcription or translation of reporterproteins, but just by detecting the disruption of the bond between plasmid DNA and a repressorprotein.

Successful Expression and purification of functional sfGFP-tagged repressorproteins in vitro

The repressor for arsenic and the Repressor of the blc operon, as well as our model protein LacI were tagged with a sfGFP c-terminally. Their spectra were characterized and their functionality could be proved by EMSA. LacI-sfGFP and arsR showed a clear EMSA shift.

Successful immobilization of DNA on paper

Based on a method proposed by Araújo et al. (Araújo et al., 2012) DNA was immobilized on Whatman Filter paper previously activated wth p-phenylene-diisothiocyanate. We made some adaptations in order to immobilise dsDNA instead of ssDNA.

Successful immobilization of Protein on paper

We pursued our second approach with was based on repressors fused to a cellulose binding domain (BBa_K1321340). All constructs were cloned successfully. When pipetted into

Successful simultaneous visualization of Protein and DNA on paper

Since the repressorproteins we wanted to detect were tagged with sfGFP, they were detectable via fluorescence. DNA was labeled with Cy3 containing primers, therefore it was also detectable on paper. Both components were visualized with the Ettan Dige. The exposure time was optimized as was the paper that was used.

References

Araújo et al., 2012, Activated Paper Surfaces for the rapid hybridization of DNA through Capillary Transport