Difference between revisions of "Team:Bielefeld-CeBiTec/Project/PRIA"
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
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| − | <p>Our second approach to cell free detection of analytes is an approach based on an | + | <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> |
| − | 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 GMOs into the wild. | + | 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. |
</p> | </p> | ||
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<h2>References</h2> | <h2>References</h2> | ||
| − | + | <p id="Araújo2012">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</p> | |
| − | < | + | |
| − | + | <p id="LehtiöJ2001">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</p> | |
| − | + | ||
| − | + | <p id="Siddiki2011"> | |
| + | Siddiki, Mohammad Shohel Rana; Kawakami, Yasunari; Ueda, Shunsaku; Maeda, Isamu (2011): Solid Phase Biosensors for Arsenic or Cadmium Composed of A <i>trans</i> Factor and <i>cis</i> Element Complex.In Sensors 2011, 11, pp. 10063-10073. 10.3390/s111110063 </p> | ||
<div class="row footer"> | <div class="row footer"> | ||
Revision as of 19:56, 9 September 2015
Plasmid Repressor Interaction Assay
A cell free detection system based on two purified components
Introduction
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. 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.
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.
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 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
Immobilized Repressor
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.
Immobilized DNA
Since the immobilization on ssDNA onto paper has been reported before (Araújo et al.) 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 et al.)
Outview
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.
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