The main achievement in this subproject was the establishment of a protocol for the Plasmid Repressor Interaction Assay (PRIA). We demonstrated that the detection of analytes in drinking water is feasible in a biological system without transcription or translation of reporter proteins, but just by detecting the disruption of the bond between plasmid DNA and a repressor protein. We optimized the system for the LacI-lacO model system and are confidential to apply it to all sorts of analytes in the near future.
The procedure performed for the PRIA was based on histagged repressor proteins immobilized on a Ni-NTA agarose column. Plasmid DNA containing the operator site for specific binding of the repressor was added to the column, unbound plasmid was washed away and the restant plasmid could be eluted by addition of the analyte to the washing buffer. We analyzed this with simple gel electrophoresis. On a future test strip the immobilized DNA-protein complex would be provided.
We worked out optimal conditions for the elution of the highest amount of DNA possible in the first elution step, since this would correspond to a high signal on the test strip. The concentration of salt turned out to be essential for the process and the optimal concentration was determined to be 500 mM potassium chloride in a slightly buffered solution. Tap water with the analyte alone could not disrupt the unspecific binding of the plasmid to the Ni-NTA column. The user would thus be required to add a certain amount of salt to his sample. Common sodium chloride is suitable for that purpose, as we tested. To further reduce the time for the assay we also tested whether the complex could be formed prior to addition of the complex to the column. In this case the bond between repressor and plasmid could not be disrupted at all. In the agarose gels DNA was visible after the elution of the protein that was performed to verify the presence of the protein at the end of the essay. This step was necessary to demonstrate that the appearance of DNA in the agarose gel was not due to the elution of the whole plasmid repressor complex.

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. To accomplish this, aminolabeled DNA is required. We made some adaptations in order to immobilize dsDNA instead of ssDNA. The original protocol demanded an 0.5 hs washing step with 4x SSC buffer, which we omitted, since this serves for the denaturing of DNA. Furthermore to be able to quantify the DNA immobilized on paper we hybridized the amino labeled operatorstrand with the complementary strand that was Cy3 labeled. This was performed via a simple annealing of two primers. So by detecting the Cy3 label via the Typhoon Fluorescence scanner we could be sure, that the immobilized DNA was the correct dsDNA.
We were not able to acquire molecular sieves at the start of our project, so we dissolved the p-phenylene diisothiocyanate (PDITC) in pure ethanol, which is easily available in any standard molecular biology laboratory. We compared the loss of the Cy3 fluorescence signal upon washing on paper that was activated with PDITC disolved in ethanol to the loss of signal upon washing on paper that was activated with PDITC disolved in dried DMSO. The loss of signal can be mainly attributed to the washing out of DNA, since an control showed, that the decrease of fluorescence of the Cy3 dye due to repeated scanning is minimal. We tried washing with three different liquids: an antibody stripping buffer, that is normally used to disrupt all protein protein interactions in an western blot; water and the binding buffer, that was normally used in our Plasmid Repressor Interaction Assay. Compared to the restant fluorescence signal on the acitvated papers the signal on the not activated papers decreases strongly, indicating that the immobilization was successful, because the signal. The washing with antibody stripping buffer resulted in blurring of the spots.

Wash with binding buffer

Wash with water