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 his-tagged repressor proteins immobilized on Ni-NTA agarose. Plasmid DNA containing the operator site for specific binding of the repressor was added to the Ni-NTA agarose, unbound plasmid was washed out and the remained plasmid could be eluted by addition of the analyte to the wash buffer. After elutioon of the plasmid the protein was eluted with an imidazole buffer. This imidazole step was to prove that the loss of protein during the procedure was not significant. We analyzed the amount of released DNA in the supernatant 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. At this concentration most DNA was eluted in the first step, which was not the case with lower concentrations. Furthermore, the binding of the plasmid to the protein was not impaired, as observed for higher concentrations.

Tap water with the analyte alone could not disrupt the binding of the plasmid to the protein. 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 (See left). Nevertheless, the solution should be slightly buffered with TRIS-HCl or sodium/potassium phosphate.
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 Ni-NTA agarose. In this case the bond between repressor and plasmid could not be disrupted at all. In the agarose gel DNA was visible after the elution of the protein with imidazole that was performed to verify the presence of the protein at the end of the assay. Furthermore, it was still seen on the Ni-NTA agarose.

We aimed at the development of a paper-based system and had optimized the procedure for immobilized protein. So we pursued the approach which was based on repressors fused to a cellulose binding domain (BBa_K1321340). We cloned and expressed fusion proteins for our LacI-lacO model system, the repressor ArsR for the detection of arsenic and for the protein BlcR, the repressor of the blc-operon, which we were working with in order to detect date rape drugs. When we pipetted the proteins onto various types of paper, their presence could be confirmed by staining and destaining of the paper with Coomassie brilliant blue and destaining solution used for SDS-PAGEs. The binding was unspecific. Any protein could be pipetted onto paper and be detected with Coomassie brilliant blue after overnight washing. Besides, most CBDs bind to microcrystalline cellulose or cotton(Lethiö et al.,2001). We were not able to find a hint about the binding of CBDs to common paper. That is why we focused on the second approach with immobilized DNA.

Washing with binding buffer
Washing with water

Based on a method proposed by Araújo et al., 2012 DNA was immobilized on filter paper previously activated with p-phenylene-diisothiocyanate (PDITC). To accomplish this, amino-labeled DNA is required. We made some adaptations to the original protocol in order to immobilize dsDNA instead of ssDNA. We omitted a 30 min washing step with 4x SSC buffer, since this serves for the denaturing of DNA. Furthermore, we hybridized the amino-labeled operator strand with the complementary strand that was Cy3 labeled to be able to quantify the DNA immobilized on paper. This was performed via a simple annealing of two oligonucleotides. 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 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 dissolved in ethanol to the loss of signal upon washing on paper that was activated with PDITC dissolved in dried DMSO. The immobilization works even better if ethanol is used as a solvent for the PDITC. The loss of signal can be mainly attributed to the washing out of DNA, since a control showed that the decrease of fluorescence of the Cy3 dye due to repeated scanning is minimal. We applied three different liquids for washing: an antibody stripping buffer, that is normally used to disrupt all protein-protein interactions in a western blot, which resulted in strong blurring of the DNA spots; water and the binding buffer that was normally used in PRIA. Compared to the remaining fluorescence signal on the activated papers the signal on the not activated paper decreases strongly, indicating that the immobilization of DNA on activated paper was successful.