Difference between revisions of "Part:BBa K1675003"
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<partinfo>BBa_K1675003 short</partinfo> | <partinfo>BBa_K1675003 short</partinfo> | ||
| − | The gene GadA (glutamate decarboxylase A) can be cloned from Escherichia coli str. K-12 substr. GadA is a functional gene for producing alkali. It catalyzes the proton-consuming conversion of glutamate to γ-amino butyric acid (GABA) whereas it converts glutamate to GABA by removing the α-carboxylate group of glutamate. | + | The gene ''GadA'' (glutamate decarboxylase A) can be cloned from ''Escherichia coli str. K-12 substr.'' ''GadA'' is a functional gene for producing alkali. It catalyzes the proton-consuming conversion of glutamate to γ-amino butyric acid (GABA) whereas it converts glutamate to GABA by removing the α-carboxylate group of glutamate. |
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| + | The following picture(Fig.1) shows the entire process of gad system. | ||
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| + | [[File:BIT_China_Regulation_System_pic3.png]] | ||
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| + | Fig.1 The mechanism of ''gadA'' | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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| + | Firstly, we cloned the gene ''GadA'' from the genome of ''Escherichia coli str. K-12.''. It is found that there are two restriction sites (EcoRI and PstI) in the sequence of ''GadA''. Thus, one-day step mutation was applied to move the restriction sites. Then, the standard part ''GadA'' was constructed successfully. | ||
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| + | In order to test the function of ''GadA'', we inserted it into plasmid pET-28a. The recombinant plasmid was transformed into BL21(DE3). The agarose gel electrophoresis analysis below (Fig.2) shows the positive clones of ''GadA''. | ||
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| + | [[File:BIT_China_Regulation_System_pic23.png]] | ||
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| + | Fig.2 The positive clones of ''GadA'' connected with pET28a | ||
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| + | After the construction of the alkali producing system, we measured the changes of pH in bacteria solution as well as the intensity of proteins expressed through SDS-PAGE. So long as OD600 of the bacterium solution reaches 0.6, the bacteria was induced by IPTG under 16℃ for 16h. The following table (Table.1) shows the different pH values between the testing group and the control group. It shows that the gene ''GadA'' does work as we expected. | ||
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| + | [[File:Example.jpg]] | ||
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| + | Table1:pH value of experimental group and control group | ||
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Revision as of 21:53, 18 September 2015
glutamate decarboxylase A （GadA）
The gene GadA (glutamate decarboxylase A) can be cloned from Escherichia coli str. K-12 substr. GadA is a functional gene for producing alkali. It catalyzes the proton-consuming conversion of glutamate to γ-amino butyric acid (GABA) whereas it converts glutamate to GABA by removing the α-carboxylate group of glutamate.
The following picture(Fig.1) shows the entire process of gad system.
Fig.1 The mechanism of gadA
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 640
Illegal AgeI site found at 700 - 1000COMPATIBLE WITH RFC[1000]