Team:Bielefeld-CeBiTec/Results/DateRapeDrugs
Date Rape Drugs
Results
Date rape drug sensor
Motivation and Experimental setup
We identified the repressor protein BlcR and its corresponding operator sequence from the blc-operon in Agrobacterium tumefaciens as a potential biosensor basis for the detection of the date rape drug ingredients γ-hydroxybutyric acid (GHB) and γ-butyrolactone (GBL). First of all, we wanted to verify the binding capabilities of the BlcR repressor by the use of EMSA-shifts. Then we tried our designed sensor system in vivo. To do so we previously needed to test the E. coli strains resistance against the both date rape drug ingredients GHB und GHL (further referred to as analytes). Then we transformed the cells with our sensor, supplemented the media with the analytes and measured the increase of the fluorescence. As the final goal of our project was to create a cell-free biosensor, in vitro tests followed.
For in vivo characterization, our genetic approach was as follows: We built a device, which contains the blc-operator sequence upstream of the translation enhancing 5’-UTR (BBa_K1758100) and sfGFP. The whole complex is under the control of the T7 Promoter. The plasmid further exhibits the BlcR repressor under the control of a constitutive promoter. Hence, the repressor is expressed, binds to the operator sequence and prevents the expression of the sfGFP. With no analyte present, the system should not give a fluorescence output signal. With an analyte present, the binding of the repressor should be weakened and a fluorescence output signal should occur.
For in vitro characterization, we transformed cells with a plasmid that carried the repressor only (BBa_K1758370). Afterwards, we prepared cell extract from this culture. Together with a reporter plasmid containing the blc-operator sequence in front of sfGFP (BBa_K1758376), we employed it in our established CFPS system.
Characterization – BlcR function
We performed EMSA-shifts and verified: BlcR binds to the operator site described in Pan et al. 2013, even when it is N-terminal fused to sfGFP (see PRIA results).
Characterization – in vivo experiments
With this proof of functionality, we set out to investigate how the two substrates (in the following referred to as analytes) GBL and GHB can influence the binding interaction.
To do so, we previously needed to test the cells resistance against GBL and GHB. Both are toxic to E. coli if their concentration in the medium exceeds a certain limit. We observed that for GHB the tolerable dose is under 1% (v/v), whereas E. coli can live in medium supplemented with 3% (v/v) GBL.
An E. coli strain carrying BBa_K1758377 in pSB1C3 (as described previously) was induced to express T7 polymerase in medium with different concentrations of either GBL or GHB. As control, medium without GBL nor GHB was used. As the strain constitutively expresses BlcR, we expected the fluorescence signal to be higher when GBL or GHB were present in the medium, as both analytes interact with BlcR. Supplementation with GHB or GBL lead to releasing of the repressor from the blc-operator, thereby raising the expression of sfGFP.
As illustrated in the nearby figure, fluorescence signals of strains that had grown in medium with the analytes were slightly higher, except for cultures with 1% GHB which showed inhibited growth.
These results indicated that, although a difference could be seen, the device has its limits in vivo.
Characterization – in vitro experiments
Because of the the issues regarding the in vivo characterization, we testes the sensor system in our CFPS-approach as described previously. We conducted a CFPS with extract from strain constitutivly expressing BlcR. As reporter plasmid, BBa_K1758376 (see figure genetic approach) was used. This plasmid is analog to our CFPS positive control PT7-UTR-sfGFP (see CFPS results) except that T7 promoter is followed by the blc-operator.
As well as in vivo, GBL and GHB had detrimental effects on the molecular machinery. 0.3% (v/v) of GBL were sufficient to strongly, but not completely inhibit protein synthesis when we used our standard cell extract. For GHB the effect was even greater, stopping protein synthesis completely at 3% (v/v) final concentration as depicted in the graphs.
The results of the CFPS reaction surpassed our expectations. In vivo, BlcR reacts on GHB and GBL and thereby dissociates from the blc-operator (Chai et al. 2007). This effect could be observed when 0.3% GBL was present in the reaction, as the flurescence signal was greater when compared to the reaction without GBL. Still, for higher concentrations of GBL, protein synthesis was inhibited.
GHB also negatively affected protein synthesis in the BlcR containing extract. Strikingly however, detrimental effects were far smaller than in the standard extract! Interestingly for 3 % GHB, the fluorescence signal surpassed the 1 % GHB signal. We propose two reasons that lead to this effect.(i) When BlcR binds to GHB, GHB is removed from the reaction and cannot act detrimentally on the molecular machinery. (ii) The polymerase is no longer blocked by BlcR which means sfGFP can be expressed.
When we normalized the signals from BlcR containing extract on our standard extract in which GHB was strongly inhibiting, the effect of BlcR was apparant immediately. Consistent with findings from Chai et al. 2007, BlcR reaction on GHB is stronger than on GBL.
We therefore demonstrated that we can detect GHB at concentrations of 1% and 3% by normalizing the fluorescence signal to a control reaction. As our CFPS system is very robust even at ethanol concentrations of 5%, we can say that we built a cell-free sensor for GHB that can be used to detect the noxious substance in liquids.
In the final application, the potential of our sensor became evident. In our paper-based CFPS reaction, water that contained 1% (v/v) GHB was used for rehydration. Fluorescence signals were measured, data quickly evaluated and our app demonstrated that the water was contaminated with date rape drugs ingredients. For detail see our final sensor approach.
Detection of γ-aminobutyrate
When we started discussing the detection of date rape drugs, we were not sure if it would be possible to detect substances such as GBL and GHB directly due to legal restrictions as well as questions with regard to dual use. Therefore, we looked for an alternative to the direct detection of date rape drugs and came across γ-aminobutyrate (GABA), which is a structural analogue of GBL. By enzymatically converting GBL to GABA, it would be possible to use a GABA biosensor for the detection of date rape drugs as well.
Literature describes operons that can be induced by GABA in both Bacillus subtilis and Rhizobium leguminosarum. We obtained the genes for the responsible proteins and the inducible promoters by gene synthesis and placed mRFP1 under the control of the inducible promoters. After growing overnight cultures with 10 mg/L GABA, we observed that the cell pellets of the B. subtilis sensor were bright red, while the pellet of the R. leguminosarum sensor did not differ from the negative control. Consequently, we decided to work with the B. subtilis sensor. Upon further characterization of the biosensor, we noticed that the background signal in LB medium was very high, possibly because it contains traces of GABA. The background signal in M9 medium was considerably lower and we observed a clear induction by GABA when growing overnight cultures with different GABA concentrations and measuring the RFP fluorescence in a plate reader. A reaction was observable down to concentrations of 1 mg/L.
We also tested whether it is possible to induce the biosensor with GBL. However, we observed no signal with three different GBL concentrations. In contrast, a qRT-PCR showed an upregulation of the gabT gene in B. subtilis. This gene is usually activated by the transcription factor GabR in the presence of GABA. We assume that GBL was metabolized to GABA in B. subtilis, which resulted in an activation of GabR. With regard to our biosensor, this confirms that the GABA sensor can be used to detect date rape drugs in combination with an enzymatic conversion.
Conclusion
We successfully built a biosensor for the detection of the common date rape drugs ingredients GHB and GBL, which is based on the BlcR repressor. A future aim for our GHB sensor would be the optimization of the BlcR containing E. coli cell extract as well as tests with purified BlcR. A higher fluorescence output would make the detection with our measurement prototype and app easier. In addition, we developed a functional biosensor for GABA, which enables an indirect detection of GHB as an alternative approach.
References
Chai, Yunrong; Tsai, Ching Sung; Cho, Hongbaek; Winans, Stephen C. (2007): Reconstitution of the biochemical activities of the AttJ repressor and the AttK, AttL, and AttM catabolic enzymes of Agrobacterium tumefaciens. In: Journal of bacteriology 189 (9), S. 3674–3679. DOI: 10.1128/JB.01274-06.
Pan, Yi; Wang, Yi; Fuqua, Clay; Chen, Lingling (2013): In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens. In: Microbiology (Reading, England) 159 (Pt 4), S. 814–822. DOI: 10.1099/mic.0.065680-0.
