Cell-free protein synthesis is a promising approach for our biosensors. However, due to the need for transcription and translation it takes up to one hour as a signal can be detected. Furthermore, the complex machinery appeared to be prone to disturbances to us. Therefore, we decided to test alternative designs. By stripping away protein synthesis and working with purfied proteins and DNA, we developed a simplified biosensor that has the potential to be very fast. The short time span is of significant advantage for biosensors in the open field, e.g. in testing beverages for date rape drugs.

In this assay, a repressor protein forms a complex with a plasmid containing its operator sequence. The repressor changes its conformation upon binding of the analyte and the bond to the DNA is disrupted. The disruption of this bond will be detected via a loss of a fluorescence signal caused by elution of labeled protein or DNA. We call this system Plasmid Repressor Interaction Assay (PRIA).
In comparison to the cell extract this method does neither require transcription nor translation of a reporter protein. Therefore, the signal can be measured much faster than in cell extracts and cellular systems. Another advantage is that microorganisms are susceptible to toxic substances, a cell-free system is supposed to tolerate a higher amount of these substances. According to Siddiki et al., who worked with a similar system based on immobilized DNA and GFP-fused repressor, measurement of arsenic and cadmium contamination in water was possible within 15 to 30 minutes (Siddiki et al. 2011), thereby meeting one of the requirements often mentioned by potential users in our survey on synthetic biology and biosensors: speed.

Furthermore, PRIA works with just two purified components, thereby minimizing the risk of releasing genetically modified organisms (GMOs) into the environment. Our system is not even subject to the regulations of the German "Gentechnikgesetz" (Genetic engineering Act). This system should be robust against environmental factors, as long as they do not denaturize the protein and show increased stability and durability compared to conventional biosensors, since there are no metabolic pathways involved (Interview with Dr. Mathias Keller, district government of East Westphalia Lippe).

Immobilized Repressor

The approach was built on fusion proteins of the repressor proteins with cellulose-binding domains (CBD). By adding these protein to paper, the repressor was immobilized. DNA which is fluorophore-labeled (in our case: Cy3-labeled) was added to the proteins. The repressor would bind the DNA on the operator site. After analyte addition, DNA was released and the loss of signal could be detected (see image).

Immobilized DNA

The immobilization on ssDNA on paper has been reported before (Araújo et al. 2012). We adapted this method to enable the immobilization of dsDNA and combined it with an approach based on the measurement of the disruption of the binding of a GFP-tagged repressor protein to immobilized DNA as reported before (Siddiki et al. 2011). In this strategy we added GFP-fused repressor protein which would bind to the immobilized DNA. After analyte addition the bond between operator DNA and GFP-fused protein would be interrupted and the signal of GFP-fused protein would be weaker after the elution step.

Outview

In summary, cell-free systems are universally usable, less sensible to toxic substances. In comparison to cellular systems, the signals are faster to detect. Moreover, they fulfil the need of biosafety. So we wanted to create a system based on repressor protein-operator DNA interaction. First, we established the proof of concept for PRIA on Ni-NTA agarose with our model system lacO-LacI. Then we wanted to show that the immobilization of dsDNA on paper is feasible. Paper-based systems become more popular due to their easiness to use and the low cost. We persued two strategies: one is based on GFP-fused repressor protein and immobilized DNA on paper, the other one is based on fluorophore-tagged DNA and immobilized repressor protein tagged to a cellulose-binding domain. In the results we implemented this assay on Ni-NTA agarose and made first steps to an immobilization of this assay on paper.

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

Santillan, M.; Mackey, M. C. (2008): Quantitative approaches to the study of bistability in the lac operon of Escherichia coli. In Journal of The Royal Society Interface 5 (Suppl_1), pp. S29-S39. DOI: 10.1098/rsif.2008.0086.focus.

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

Results

And what did we detect with these systems?