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.
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.
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
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