Difference between revisions of "Part:BBa K1758361"

Latest revision as of 05:53, 19 September 2015

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.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.

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.

@@ Line 19: / Line 19: @@
 *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.
+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
-[[File:Bielefeld-CeBiTec_gabR_induction_GABA.png|700px|thumb|center|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.]]
+===Characterization===
+We measured the response of this device to differnt concentrations of GABA. The experiment was performed as follows:
+*Single colonies of <i>E. coli</i> 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 OD<sub>600</sub> 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 OD<sub>600</sub> was measured with 200 µL in a transparent 96 well plate.
+*The values were normalized as follows: The OD<sub>600</sub> of M9 medium was subtracted from the OD<sub>600</sub> 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.
+[[File:Bielefeld-CeBiTec_2015_GABA_induction_small.png|700px|thumb|center|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.
-[[File:Bielefeld-CeBiTec_2015_GABA_GBLeffect_small.png|700px|thumb|center|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 OD<sub>600</sub> of M9 medium was subtracted from the OD<sub>600</sub> 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.
+[[File:Bielefeld-CeBiTec_2015_GABA_GBLeffect_small.png|600px|thumb|center|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.]]