Part:BBa_K1758377
Biosensor device for detection of GHB and GBL
Overview
GHB (γ-hydroxybutyrate) and GBL (γ-butyrolactone) are chemicals misused by criminals as date rape drug ingredients (for details please be referred to our wiki). We found an inducible operon in Agrobacterium tumefaciens that was perfectly suited for building a biosensor to detect theses substances.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 981
Illegal NheI site found at 1004
Illegal NheI site found at 1786 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1494
Illegal NgoMIV site found at 1735 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 122
Catabolism of γ-butyrolactone (GBL) in A. tumefaciens
The Blc-Operon and the catabolism of GBL in A. tumefaciens. GHB: γ-hydroxybutyrate; GBL: γ-butyrolactone; SSA: Succinic semialdehyde; SA: Succinate.
The plant pathogen Agrobacterium tumefaciens has been widely used in plant research, as it can transform plant cells with the help of the Ti-plasmid (tumor-inducing plasmid). A. tumefaciens is able to utilize the uncommon carbon and energy source γ-butyrolactone (GBL) found in plants. To do this, the bacterium uses the enzymes of the blcABC operon (Chai et al. 2007). This operon is localized on the second megaplasmid of A. tumefaciens C58 strain, pAtC58. blcABC expression is controlled by the repressor protein BlcR . When GBL is not present, two dimers of BlcR form a tetramer and inhibit operon expression by binding to an operator sequence localized in front of the operon, thereby hindering the polymerase from transcribing the DNA (Pan et al. 2011, 2013). The inhibition of operon repression was shown to be inducible not only by GBL, but even stronger by GHB (gamma-hydroxybutyrate) and SSA (succinic semialdehyde) (Chai et al. 2007).
The enzymes coded for in the blcABC operon catalyze the reaction of GHB to GBL in a first step. If GBL is taken up by an organism containing the operon, it is processed to GHB that even stronger induces the operon. Further, GHB is processed to succinic semialdehyde that finally enters the GABA pathway. Therefore, the operon enables the chassis to utilize GHB or GBL as carbon and nitrogen source. It has been shown that the transformation of the operon enables E. coli to grow on GBL as sole carbon source. (Carlier et al. 2004). Hence, as further experiments, the induction might be increased by the additional transformation of the operon containing blcA.
Molecular function of BlcR repression and derepression.
Therefore, our sensor is based on the repressor BlcR under the control of a constitutive promoter, and the binding sequence of the promoter Pblc. This binding sequence is following an inducable T7 promoter. The promoter is followed by a 5' untranslated region and controls transcription of sfGFP, which is the output signal. The device can detect both GBL and GHB.
Experimental design
First of all, we wanted to verify the binding capabilities of the BlcR repressor by the use of EMSA-shifts
For in vivo characterization of the biosensor device, this part was transformed and the cells were cultivated. With no analyte present, the system should not give a fluorescence output signal according to repressor action on the operator. 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 this part 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.
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