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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1380
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2130
  Illegal AgeI site found at 2242
  
• 1000
  COMPATIBLE 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. 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.

Induction by GABA. 5 mL cultures in M9 with different GBL concentrations were grown overnight and RFP fluorescence was measured in a plate reader. The error bars represent the standard deviation of three biological replicates.

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.

Effect of GBL on GABA sensor. 5 mL cultures in M9 with different GBL concentrations were grown overnight and RFP fluorescence was measured in a plate reader. The error bars represent the standard deviation of three biological replicates.

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.