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The enzymatic reaction was conducted in a 2 mL reaction mixture consisted of 200 mM Na2HPO4-citric acid buffer (pH4.0), 50 mM L-MSG, 0.01 mM PLP, and 50–100 μL of purified enzyme. 1-fluoro-2,4-dinitrobenzene (FDNB) was then added to the reaction to get a yellow compound dinitroxine benzodiazepines (DNP amino acids) which has an absorbance at 485 nm. The mixtures were thoroughly mixed and incubated at 60°C for 1 hour. After cooling to room temperature, added PBS (0.02mol/L, pH=7.0) to the mixtures to make a final volume of 10 mL. Then took 1.7 mL and measured the absorbance at 485 nm. The result was OD485nm = 1.16 (1.39 ng/mL of GABA), indicating that GABA was indeed generated in the reaction and the function of gadB was as expected.  
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The enzymatic reaction was conducted in a 2 mL reaction mixture consisted of 200 mM Na2HPO4-citric acid buffer (pH4.0), 50 mM L-MSG, 0.01 mM PLP, and 50–100 μL of purified enzyme. 1-fluoro-2,4-dinitrobenzene (FDNB) was then added to the reaction to get a yellow compound dinitroxine benzodiazepines (DNP amino acids) which has an absorbance at 485 nm. The mixtures were thoroughly mixed and incubated at 60°C for 1 hour. After cooling to room temperature, added PBS (0.02mol/L, pH=7.0) to the mixtures to make a final volume of 10 mL. Then we measured the absorbance at 485 nm. The result was shown below, indicating that GABA was indeed generated in the reaction and the function of gadB was as expected.  
  
 
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Revision as of 10:56, 20 October 2021


gadB

Gene gadB produces an enzyme called GadB, which can catalyze the conversion of glutamate to GABA.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 700
  • 1000
    COMPATIBLE WITH RFC[1000]


Demonstrate the function of gadB

Gene gadB was amplified using E. coli genome as the template. We used the Golden Gate Assembly method to construct the following plasmid T7-T7RBS-gadB-T500, that is the gene gadB is under the control of the classic T7 promoter and its corresponding ribosome binding site, with a terminator T500. The plasmid was transformed into E. coli BL21(DE3) for expression. After induction with 100 μM IPTG, cells were lysed and GadB proteins were purified.

Fig 1. An SDS-PAGE gel of GadB protein purification. 50mmol/L, 100 mmol/L, 200 mmol/L, 500 mmol/L were samples collected when eluted using elution buffer with 50 mM imidazole, 100 mM imidazole, 200 mM imidazole, and 500 mM imidazole, respectively.

The enzymatic reaction was conducted in a 2 mL reaction mixture consisted of 200 mM Na2HPO4-citric acid buffer (pH4.0), 50 mM L-MSG, 0.01 mM PLP, and 50–100 μL of purified enzyme. 1-fluoro-2,4-dinitrobenzene (FDNB) was then added to the reaction to get a yellow compound dinitroxine benzodiazepines (DNP amino acids) which has an absorbance at 485 nm. The mixtures were thoroughly mixed and incubated at 60°C for 1 hour. After cooling to room temperature, added PBS (0.02mol/L, pH=7.0) to the mixtures to make a final volume of 10 mL. Then we measured the absorbance at 485 nm. The result was shown below, indicating that GABA was indeed generated in the reaction and the function of gadB was as expected.

Fig 2. A standard curve of GABA.