Team:Bielefeld-CeBiTec/Results/PRIA

PRIA Results

A Cell-free Detection System based on two purified Components

Introduction

Paper-based and cell-free systems offer many advantages. They are easy to use, universally applicable and safe. As a further development of the cell-free protein synthesis (CFPS) we created an in vitro cell-free system based on the binding of a purified repressor protein to purified DNA. Our aim was to establish an alternative assay type that works in vitro on paper. Repressor protein and labeled DNA with the operator site form a complex in this assay. If the analyte is added to the complex, the repressor changes its conformation and releases the DNA. For more information about the background, read this part.
In general, there are two different strategies. In our first strategy the protein is immobilized and the DNA is added afterwards. In our second strategy the DNA is immobilized first and fluorescence tagged protein is then added. Alternatively as a modification of the strategies, the DNA-protein complex is formed in solution and immobilized subsequently.
For the application of our biosensor the immobilized DNA-protein complex would be distributed to the users, they would add their sample and a signal would be generated by the dissociation of the DNA -protein complex.
To show that our approach operates as expected, we first needed to prove that the complex formation with the repressor proteins and immobilization in vitro is reversible by the addition of the analyte. We started our experiments with the repressor of the lac operon from E. coli (LacI) and its corresponding operator site lacO. We chose this model system, because it is well characterized and the substance the repressor reacts to is isopropyl Β-D-1-thiogalactopyranoside (IPTG), which is easily available in molecular biology laboratories.

Successful Detection of an Analyte in vitro

Figure 1: General workflow of the Plasmid Repressor Interaction Assay

Figure 2: Device for the expression of LacI-His (BBa_K1758201)

Figure 3: Effect of different salt concentrations on DNA amount in the first elution step. The PRIA was performed with our model system consisting of immobilized LacI. The plasmid contained the lac operator. Agarose gels with samples from washes and elutions of each step of the PRIA after addition of the plasmid. Each PRIA was performed with a buffer containing different amounts of potassium chloride. The only difference between the wash and the elution buffer was the addition of the analyte (in this case IPTG). After the third elution step, the protein bound to the Ni-NTA agarose was eluted with a buffer containing a high concentration of imidazole to confirm the protein's presence at the end of the assay. The Ni-NTA agarose was also applied to the gel to check for remaining plasmid bound to it. The negative control was plasmid applied to Ni-NTA agarose without protein and washed with 250 mM KCl buffer. According to the tests with different salt concentrations in buffer we need to use the buffer with 500 mM salt.

Figure 4: Quantification of DNA in different steps of PRIA with PicoGreen assay, n=3. The DNA is mainly eluted after the addition of the analyte in the elution step, but not in the washing steps. This shows that our assay operates as expected.

The procedure performed for PRIA was based on his-tagged repressor proteins immobilized on Ni-NTA agarose.(For the general workflow see figure 1.) Herefore, we constructed a device for the expression of his-tagged LacI (see figure 2). After purification of the protein with the Protino® Ni-TED 1000 Packed Columns Kit from Macherey-Nagel we immobilized it on Ni-NTA agarose in a reaction tube. 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 remaining plasmid could be eluted by addition of the analyte to the wash buffer. The DNA amount eluted upon addition of the analyte depends strongly on the salt concentration in the buffer used for washing and elution (see figure 3). We analyzed the released DNA in the supernatant with agarose gel electrophoresis. The DNA amount eluted in the first elution step compared to the total DNA amount bound to the agarose after the forth washing step, is much higher (see figure 4). An explanation for an increased DNA amount in the elution steps could have been dissociation of the protein and the bound DNA from the Ni-NTA agarose. The last step of the assay is elution of the protein from the Ni-NTA agarose with an imidazole buffer to confirm the presence of the protein at the end of the procedure. Our analysis of the samples via SDS-PAGE shows clearly, that there is nearly no loss of protein during the assay and most of the protein can be recovered upon elution with imidazole (see figure 5). On a future test strip the immobilized DNA-protein complex would be provided, so the user just needs to add the potentially contaminated water plus sodium chloride and buffer solution which we would provide as a biosensor kit.

Moreover, we tested many different conditions to further evaluate the robustness of PRIA. Tap water with the analyte alone could not disrupt the binding between the plasmid and the protein, the major part of plasmid remains bound to the protein. In an agarose gel DNA can be detected in the steps where the protein (and the DNA bound to it) is eluted and in the fraction with the rest of the Ni-NTA agarose, which also still contains protein-DNA complexes (see figure 6). Thus, the user would be required to add a certain amount of salt to the sample. In our assay we mainly applied potassium chloride, but common sodium chloride is suitable for that purpose, too (see figure 7). 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. The results were similar to the performance with water instead of buffer. 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. This was performed to verify the presence of the protein at the end of the assay. Furthermore, DNA could still be detected on the Ni-NTA agarose (see figure 8).

Figure 5: Test for loss of protein in different steps of PRIA

Figure 6: PRIA performed with water instead of buffer

Figure 7: Usage of 500 mM NaCl instead of 500 mM KCl in binding buffer

Figure 8: PRIA with DNA-protein complex added to Ni-NTA agarose

Our Strategies on Paper

After having created a functional assay with lacO-LacI, our next step was to implement this approach immobilized on paper. To achieve this, we pursued two strategies. For the first strategy we immobilized repressor proteins fused with a cellulose binding domain from from Imperial 2014 (BBa_1321340) on paper (see figure 9). After adding Cy3-labeled DNA with the operator site, the complex formation should be reversed through the analytes. Herefore, we we constructed devices for fusion proteins of cellulose binding domain (CBD) with LacI for a proof of concept, BlcR (the repressor protein for our biosensor detecting date rape drugs) and ArsR (the repressor for the biosensor detecting arsenic) for a real world application.
Our second strategy is based on immobilized DNA which has a Cy3- and an amino-label (We also labeled the DNA with Cy3 for verifying that DNA is immobilized and is not washed out, but generally the signal in this strategy is generated with sfGFP tagged repressors.). This time we fused super folder GFP (sfGFP) to the same repressors (BBa_K1758202, BBa_K1758203, BBa_K1758204) and wanted to detect the released protein after analyte addition (see figure 10). The DNA containing the sequences of arsO (BBa_J33201), P_blc (BBa_K1758375) and lacO were applied as plasmid DNA for binding the corresponding repressor. How we immobilized protein or DNA on paper is further described in the next sections.

Figure 9: Composition of the fusion proteins with CBD

Figure 10: Composition of the fusion proteins with sfGFP

Immobilization of Protein on Paper

Figure 11: Cell lysate containing repressor proteins LacI and BlcR fused to a cellulose binding domain or sfGFP immobilized on paper and stained with Coomassie Brilliant Blue after overnight washing

We aimed at the development of a paper-based system and optimized the procedure for immobilized protein. We worked with the approach which was based on repressors fused to a cellulose binding domain (BBa_1321340) first. We cloned and expressed fusion proteins for our LacI-lacO model system (BBa_K1758202), the repressor ArsR (BBa_K1758203) for the detection of arsenic and for the protein BlcR, the repressor of the blc-operon (BBa_K1758204), which we were working with in order to detect date rape drugs. When we added the proteins on various types of paper, their presence could be confirmed by staining the paper with Coomassie brilliant blue and destaining with destaining solution used for SDS-PAGEs. The binding was unspecific, since the spots with proteins containing the CBD and the spots containing proteins fused with sfGFP, which is not supposed to bind to paper specifically, still showed the same intensity of blue dye after overnight washing (see figure 11). Besides, most CBDs bind to microcrystalline cellulose or cotton (Lethiö et al.). We were not able to find a hint about the binding of CBDs to common paper. Additionally, we came across certain difficulties concerning the purification of proteins with a CBD. That is why we focused on the second strategy with immobilized DNA on paper.

Successful Immobilization of DNA on Paper

Figure 12: 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., 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 the paper. This was performed via a simple annealing of these two oligonucleotides. Therefore, by detecting the Cy3 label via the Typhoon fluorescence scanner, we could be sure that the immobilized DNA was the correct dsDNA.

We dissolved PDITC, the substance for activation of paper for DNA immobilization, in pure ethanol and DMSO. We compared which solvent is better for the activation. Herefore, we washed the immobilized DNA on paper, which was activated differently, and compared the remaining intensities of the Cy3 label on paper after washing (see figure 12). 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:

1. an antibody stripping buffer that is normally used to disrupt all protein-protein interactions in a western blot. This resulted in strong blurring of the DNA spots.
2. water
3. the binding buffer that was normally used in PRIA

Compared to the remaining fluorescence signal on the activated papers the signal on the unactivated paper decreases strongly (see figures 13 and 14). This indicated, that the immobilization of DNA on activated paper was successful. The fluorescence signals of the scanned gels were quantified with the image processing software ImageJ.

Washing with binding buffer

Figure 13: 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 fluorescence scanner 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

Figure 14: 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 of 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 (BBa_K1758203), date rape drugs (BBa_K1758204), as well as our model protein LacI C-terminally fused to sfGFP (BBa_K1758202). Their specific binding to the operator DNA could be proven by electrophoretic mobility shift assay (EMSA). Upon increasing amounts of protein binding to the DNA the electrophoretic mobility of the DNA fragments decreases. This results in a shift between protein-DNA complexes and free DNA. It is visible that the labeled operator sites without protein added to them run faster in the gel compared to the operator sites occupied by the corresponding binding proteins (see figure 15). So all purified fusion proteins retained their DNA-binding function. The two different shifts in the LacI-sfGFP EMSA result from the formation of tetramers which is typical for LacI. In some cases dithiothreitol (DTT) was added to the reaction. It simulates reducing conditions, which are normally present in the cell. This can influence the performance of the repressors. In the case of BlcR it had no effect, but it enhances the binding of LacI-sfGFP to its corresponding binding site.

Figure 15: EMSA shift caused by addition of different amounts of the indicated protein to 0.05 pmol (0.5 pmol for ArsR-sfGFP) Cy3-labeled operator site. +/- refers to the presence of dithiothreitol in the reaction.

Successful simultaneous Visualization of Protein and DNA on Paper

Figure 16: Scan of the Ettan Dige Fluorescence Scanner. Spots of Cy3 labeled DNA (red) and sfGFP tagged repressor proteins (green) applied to common filter paper.

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 needed a method for the simultaneous visualization of both components. Since the repressor proteins were tagged with sfGFP, they could be detected via fluorescence. Cy3-labeled DNA was also detectable via fluorescence (at different extinction and emission wavelengths to sfGFP) on paper. We visualized both components with an Ettan Dige Scanner normally used for 2D-SDS-PAGEs (see figure 16). The exposure time and the applied paper were optimized. The filter paper used was Schleicher & Schuell filter paper (Ref. No. 311 609), but several types of paper were suitable for this application. The optimal exposure times were 150 ms for the Cy3 and the Cy2 channel (Cy2 channel for measuring the fluorescence signal of GFP).

Fluorescence Measurements in Plate Reader

Figure 17: Change in fluorescence upon addition of arsenic in a 96-well plate coated with cellulose and with immobilized DNA. Different conditions were tested.

As a first approach to check whether the formation and dissociation of the complex was measurable and quantifiable, we decided to adapt a method for the examination of in vitro complex formation and dissociation described earlier: 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 results with a 96 well plate coated with Avicell microcrystalline cellulose. The measurement in the Infinite M200 plate reader (Tecan) suggests that DNA was immobilized until the end of the assay because the Cy3 signal is significantly above the background signal generated by the cellulose (see figure 18). Nevertheless, the measurement of the supernatant fluorescence showed no clear tendency, neither did the fluorescence that remained in the wells. We tested different conditions in order to optimize the protocol: 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 (see figure 17). Probably this was due to the Avicell cellulose, which was not easy to handle, since large amounts of it were lost during the different washing or transfer steps.

Figure 18: Comparison of fluorescence signals at 590 nm (emission wavelength) in wells of 96 well plate at the end of the assay. Cy3 labeled DNA can be detected at 590 nm. n=3 (technical replicates)

Conclusion

In summary, PRIA has the potential to become a robust real world application as a novel biosensor. We delivered a convincing proof of concept for the detection of an analyte based solely on in vitro interaction of protein and DNA. Nevertheless, buffers need to be optimized and adjusted to the specific proteins. The assay is functional on Ni-NTA agarose and only takes 15 min for the generation of a signal. Applying the method to paper will be a challenge, since the unspecific absorption of the protein by the paper has to be avoided in order to be able to establish a stable protein-DNA complex. Our results concerning the immobilization of DNA are promising, the DNA is bound with high stability on common filter paper and can be washed for at least 45 min without significant signal loss. In future applications, PRIA can be adapted to detect several substances, for example heavy metals.

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 the analytes for our biosensors apart from IPTG?