Difference between revisions of "Part:BBa K1675003"
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Fig.1 The mechanism of ''gadA'' | Fig.1 The mechanism of ''gadA'' | ||
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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. | 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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Fig.2 The positive clones of ''GadA'' connected with pET28a | Fig.2 The positive clones of ''GadA'' connected with pET28a | ||
| − | 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. | + | 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. |
| − | [[File: | + | [[File:pH.png]] |
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| + | Table.1: pH value of experimental group and control group | ||
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| + | <!-- Add more about the biology of this part here | ||
| + | ===Usage and Biology=== | ||
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Latest revision as of 22:00, 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
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.
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.
Fig.2 The positive clones of GadA connected with pET28a
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.
Table.1: pH value of experimental group and control group
Sequence and Features
- 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]