Revision as of 02:33, 24 August 2015

iGEM Bielefeld 2015

PRIA-Results

A cell free detection system based on purified components

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.

Immobilization of Protein on paper

We pursued our second approach which was based on repressors fused to a cellulose binding domain (BBa_K1321340). All constructs were cloned successfully. When pipetted onto various types of paper, its presence could be confirmed by staining and destaining of the paper with Coomassie brilliant blue and destaining solution used for SDS-PAGES (45 % EtOH, 10 % acetic acid in ddH₂O). The binding was quite unspecific though, because most CBDs just bind to microcristalline cellulose or cotton, but not to common paper. That is why we focused on the approach with immobilized DNA.

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.

Tecan Measurements

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References

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

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            <h2>Immobilization of Protein on paper </h2>
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-        <div><p> We pursued our second approach which was based on repressors fused to a cellulose binding domain (<a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a>). All constructs were cloned successfully. When pipetted onto various types of paper, its presence could be confirmed by staining and destaining of the paper with Coomassie brilliant blue and destaining solution used for SDS-PAGES (45 % EtOH, 10 % acetic acid in ddH<sub>2</sub>O). The binding was quite unspecific though, because most CBDs just bind to microcristalline cellulose or cotton, but not to common paper. That is why we focused on the approach with immobilized DNA.
+        <div><p> We pursued our second approach which was based on repressors fused to a cellulose binding domain (<a href="http://parts.igem.org/Part:BBa_K1321340" target="_blank">BBa_K1321340</a>). All constructs were cloned successfully. When pipetted onto various types of paper, its presence could be confirmed by staining and destaining of the paper with Coomassie brilliant blue and destaining solution used for SDS-PAGES (45 % EtOH, 10 % acetic acid in ddH<sub>2</sub>O). The binding was quite unspecific though, because most CBDs just bind to microcristalline cellulose or cotton, but not to common paper. That is why we focused on the approach with immobilized DNA.
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