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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.
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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, indicating that GABA was indeed generated in the reaction and the function of gadB was as expected.
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        <img src="https://2021.igem.org/wiki/images/a/ac/T--FZU-China--resul-10.jpg" width="50%" style="float:center">
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Fig 2. A standard curve of GABA.
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Revision as of 16:29, 18 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 took 1.7 mL and measured the absorbance at 485 nm. The result was OD485nm = 1.16, indicating that GABA was indeed generated in the reaction and the function of gadB was as expected.

Fig 2. A standard curve of GABA.