Team:Bielefeld-CeBiTec/Results/DateRapeDrugs
Date Rape Drugs
Results
Detection of GBL and GHB
We demonstrated that we can detect the date rape drug ingredients γ-butyrolactone (GBL) and γ-hydroxybutyric acid (GHB) with the help of a small protein, BlcR, in combination with its related DNA sequence, the blc-operator. We used various methods to characterize the interaction process.
We performed EMSA and verified that BlcR binds to the operator site described in Pan et al. 2013, even when it is N-terminally fused to sfGFP (see PRIA results.
With this proof of functionality, we set out to investigate how the two analytes GBL and GHB can influence the interaction.
GBL and GHB are both toxic to E. coli when 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 survive in medium with 3% (v/v) GBL.
An E. coli strain carrying BBa_K1758377 in pSB1C3 was induced to express the T7 polymerase in medium with different concentrations of either GBL or GHB. As control, medium without GBL nor GHB was used. Induction lead to expression of sfGFP. However, the sfGFP coding sequence follows on the blc operator sequence. 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 and inhibit its binding to the operator.
Fluorescence signals of strains that had grown in medium with 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. We conducted a CFPS with extract from strain constitutivly expressing BlcR. As reporter plasmid, BBa_K1758376 was used. This plasmid equals 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 influence, 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.
This however did not stop us from further testing. We took E. coli that constitutively expressed BlcR (BBa_K1758370). In less than a day we cultivated the cells, made cell extract via sonification and performed CFPS with BBa_K1758376 as reporter. In standard extract, fluorescence signals of our positive control plasmid and BBa_K1758376 were similar.
The results of the CFPS reaction surpassed all expectations. In vivo, BlcR reacts zo 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 fluorescence 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 standard extract! Especially interesting was that for 3% GHB, the fluorescence signal surpassed the 1% GHB signal. We propose two reasons that lead to this effect together: When BlcR binds to GHB, on the one hand GHB is removed from the reaction and cannot act detrimentally on the molecular machinery, and on the other hand the polymerase is no longer blocked by BlcR which means sfGFP can be expressed.
When we normalized the signals from the BlcR containing extract on our standard extract in which GHB was strongly inhibiting, the effect of BlcR could not be overlooked. Consistent with findings from Chai et al. 2007, the 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 were 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
As an alternative to the direct detection of GBL or GHB, we constructed biosensors for the structural analogue γ-aminobutyrate (GABA). By enzymatically converting GBL to GABA, it would be possible to use such a sensor for the detection of date rape drugs as well.
We found out that Bacillus subtilis and Rhizobium leguminosarum possess operons that can be induced by GABA. We obtained the genes for the responsible proteins and the inducible promoters by gene synthesis and placed mRFP1 under the control of the inducable promoters. After growing overnight cultures with 10 mg/L GABA, we observed that the cell pellets of the B. subtilis sensor were clearly 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
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.
