Difference between revisions of "Part:BBa K1758360"

Latest revision as of 21:53, 17 September 2015

gabR gene and gabT promoter from Bacillus subtilis

This is a part of the gab operon of Bacillus subtilis. It includes the gene for the transcriptional regulator GabR under the control of its natural promoter as well as the gabT promoter, which is regulated by GabR. GabR is activated by γ-aminobutyrate (GABA). The expression of any gene that is inserted downstream of this part can therefore be induced by GABA.

Sequence and Features

Usage and Biology

This part consists of the gabR gene from Bacillus subtilis and the intergenic region between gabR and gabT, which includes the promoters of both genes. 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 part in combination with a reporter gene. We characterized this part after inserting mRFP1 downstream of the part. The data show a clear induction of mRFP1 expression in the presence of GABA.

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.