Team:Bielefeld-CeBiTec/Results/PRIA

PRIA Results

A Cell-free Detection System based on two purified Components

Successful Detection of an Analyte in vitro

Effect of different salt concentrations on amount of DNA in the first elution step

Quantification of DNA in different steps of PRIA

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.

Usage of 500 mM NaCl instead of 500 mM KCl

PRIA performed with water instead of buffer

PRIA with DNA-protein complex added to Ni-NTA agarose

Immobilization of Protein on Paper

Repressor proteins LacI and BlcR fused to a cellulose binding domain or sfGFP immobilized on paper after overnight washing

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.

Successful Immobilization of DNA on Paper

Original scan from the Typhoon. On the left hand side you see the papers prior to washing, on the right hand side the same papers after 45 min of washing in different solutions.

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.

Washing with binding buffer

Development of the fluorescence signal of Cy3 labeled DNA that was immobilized on paper activated with PDITC dissolved in DMSO or ethanol respectively. Unactivated paper served as negative control. The paper was washed for 45 min with the binding buffer used in PRIA for stabilizing the protein DNA complex and scanned with the Typhoon various times during this process. The fluorescence was quantified with ImageJ and the peak areas were normalized to the values before the first wash.

Washing with water

Development of the fluorescence signal of Cy3 labeled DNA that was immobilized on paper activated with PDITC dissolved in DMSO or ethanol respectively. Unactivated paper served as negative control. The paper was washed for 45 min with water and scanned with the Typhoon various times during this process. The fluorescence was quantified with ImageJ and the peak areas were normalized to the values before the first wash.

Successful Expression and Purification of functional sfGFP-tagged Repressor Proteins

For the approach to a paper-based test strip with immobilized DNA, super folding Green Fluorescent Protein (sfGFP)-tagged repressor proteins are required for the generation of a signal upon release. We tagged the repressors for the detection of arsenic, date rape drugs, as well as our model protein LacI with sfGFP C-terminally. Their specific binding to the operator DNA could be proven by electrophoretic mobility shift assay (EMSA). Upon increasing amounts of protein binding to the electrophoretic mobility of the DNA fragments decreases. This generates the EMSA shift.(See below.)

EMSA shift caused by addition of different amounts of LacI-sfGFP to 0.05 pmol Cy3-labeled operator site.

EMSA shift caused by addition of different amounts of BlcR-sfGFP to 0.05 pmol Cy3-labeled operator site.+/- indicates whether dithiothreitol was added to the reaction.

EMSA shift caused by addition of different amounts of ArsR-sfGFP to 0.5 pmol Cy3-labeled operator site.

Successful simultaneous Visualization of Protein and DNA on Paper

Cy3 labeled DNA is shown in red, sfGFP tagged repressor proteins are displayed in green. Common filter paper was the base for this experiment.

The next step for us was to establish the DNA-protein complex on paper. In order to test if the complex formation was successful we required at first an method for the simultaneous visualization of both components. Since the repressor proteins 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. We visualized both components with an Ettan Dige Scanner normally used for 2D-SDS-Pages. The exposure time was optimized as was the paper that was used.

Fluorescence Measurements in Plate Reader

Change in fluorescence upon addition of arsenic.

Siddiki et al. immobilized biotinylated DNA in a 96-well plate and added cell lysates to each well. The cell lysates contained GFP-tagged repressor proteins. After washing the wells once they would add their sample to the wells, wait for 15-30 minutes and measure the fluorescence in the supernatant. They report a concentration-dependent increase of the fluorescence in response to cadmium and arsenic. This increase in fluorescence was due to the dissociation of the GFP-tagged repressor proteins from the immobilized operator sites: the tagged proteins were eluted from the wells and became part of the supernatant. (Siddiki et al.)
We tried to reproduce these finding with a 96 well plate coated with Avicell microcrystalline cellulose. The Tecan measurement suggests that DNA was immobilized because the Cy3 signal is significantly above the background signal generated by the cellulose. Nevertheless, the measurement of the supernatant fluorescence showed no clear tendency, neither did the fluorescence that remained in the wells. We tested different conditions: the buffers used in our optimized PRIA protocol and the buffers described by Siddiki et al., to avoid unspecific binding of the protein to the cellulose we tested the effect of blocking the wells with milk powder solution prior to addition of the cell lysate for both buffer combinations. None of the observed changes were significant.

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

What were we able to detect?