Part:BBa_K1758361
mRFP1 controlled by the GABA-inducible activator GabR
gabR encodes a transcriptional regulator which activates the expression of mRFP1 in the presence of γ-aminobutyrate (GABA). This device is consequently a GABA sensor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1380
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2130
Illegal AgeI site found at 2242 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This part consists of the gabR gene from Bacillus subtilis with its natural promoter and mRFP1 under control of the gabT promoter. gabR encodes a transcription factor which in vivo activates the expression of the gabTD operon in the presence of γ-aminobutyric acid (GABA) and pyridoxal 5′-phosphate (PLP). gabT and gabD encode γ-aminobutyrate aminotransferase and succinic semialdehyde dehydrogenase, respectively. These enzymes enable B. subtilis to utilize GABA as a nitrogen and carbon source. GabR also negatively autoregulates its own expression, both in the presence and abscence of GABA.
More information can be found here:
- Belitsky, Boris R. (2004): Bacillus subtilis GabR, a protein with DNA-binding and aminotransferase domains, is a PLP-dependent transcriptional regulator. In Journal of molecular biology 340 (4), pp. 655–664. DOI: 10.1016/j.jmb.2004.05.020.
- Belitsky, Boris R.; Sonenshein, Abraham L. (2002): GabR, a member of a novel protein family, regulates the utilization of γ-aminobutyrate in Bacillus subtilis. In Molecular Microbiology 45 (2), pp. 569–583. DOI: 10.1046/j.1365-2958.2002.03036.x.
- Wang, Wei; Kreinbring, Cheryl A. et al. (2013): Crystal structure of Bacillus subtilis GabR, an autorepressor and transcriptional activator of gabT. In Proceedings of the National Academy of Sciences of the United States of America 110 (44), pp. 17820–17825. DOI: 10.1073/pnas.1315887110.
- Uniprot entry on GabR: P94426
We worked with this part because GABA is structurally related to GHB, a frequently used date rape drug. By enzymatically converting GHB to GABA it would be possible to detect this drug using this device.7
Characterization
We measured the response of this device to differnt concentrations of GABA. The experiment was performed as follows:
- Single colonies of E. coli harboring the plasmid were obtained from the glycerol stock by plating.
- Three colonies were used to inoculate 5 mL cultures in M9 with antibiotic.
- The precultures had to be cultured for 48 h at 37 °C to reach a suitable OD.
- The cultures were diluted to an OD600 of 0.8. 300 µL were then added to 4.6 mL M9 with antibiotic and 100 µL GABA solution.
- The measurement was carried out on the following day. RFP fluorescence of 50 µL culture was measured at 570 nm and 607 nm in a black 384 well plate. The OD600 was measured with 200 µL in a transparent 96 well plate.
- The values were normalized as follows: The OD600 of M9 medium was subtracted from the OD600 of the cultures. The relative fluorescence was divided by the optical density of the culture. Finally, the normalized fluorescence of a culture without plasmid-encoded fluorescence proteins was subtracted from the other values. The mean of two cultures was used for this autofluorescence correction, because one culture did not grow. All other values were measured in triplicate.
Our data confirm that mRFP1 expression can be induced by GABA. A reaction can be observed down to concentrations of 1 mg/L. It should be noted that the measuremts were carried out in M9 medium, as we observed a strong background signal in LB.
In addition, we tested whether it is possible to directly induce our biosensor with GBL. As expected, there was no difference to the negative control.
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