Difference between revisions of "Team:Bielefeld-CeBiTec/Project/PRIA"
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Revision as of 00:47, 5 September 2015
PRIA
Plasmid-Repressor-Interaction Assay
Introduction
Our second approach to cell free detection of analytes is an approach based on an immobilized repressor that binds to a plasmid. When the analyte binds the repressor, it changes its conformation and releases the DNA. This release is to be measured. We called 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 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
...
Immobilized DNA
...
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 et al., 2012, Activated Paper Surfaces for the rapid hybridization of DNA through Capillary Transport