Difference between revisions of "Part:BBa K2547000"
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| − | This part is the coding sequence (CDS) of wild-type carbonic anhydrase (CA2) | + | <p>This part is the coding sequence (CDS) of wild-type human carbonic anhydrase 2 (CA2) with a His-tag placed at the C-terminal. CA2 is a metalloenzyme that can efficiently catalyze the formation of HCO<sub>3</sub><sup>-</sup> and H<sup>+</sup> from CO<sub>2</sub> with high catalytic efficiency and fast reaction rate. |
| − | + | </p> | |
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===Usage and Biology=== | ===Usage and Biology=== | ||
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<partinfo>BBa_K2547000 parameters</partinfo> | <partinfo>BBa_K2547000 parameters</partinfo> | ||
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| − | <p>We first synthesized the sequence of | + | <p><h3>Construction of wild-type human carbonic anhydrase 2 (CA2-WT) expression plasmid </h3>We first synthesized the sequence of CA2-WT, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig. 1 and Fig. 2). |
| + | <br></p> | ||
<div align="center"> | <div align="center"> | ||
| − | https://static.igem.org/mediawiki/parts/ | + | https://static.igem.org/mediawiki/parts/f/fe/T--AHUT_China--_zhiliopopp.jpg |
</div><br><br> | </div><br><br> | ||
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<div align="center"> | <div align="center"> | ||
https://static.igem.org/mediawiki/parts/1/1e/T--AHUT_China--_2part.jpg</div> | https://static.igem.org/mediawiki/parts/1/1e/T--AHUT_China--_2part.jpg</div> | ||
| − | <center>Fig. 2 Agarose Gel Electrophoresis of CA2-WT recombinant plasmid and its identification by enzyme digestion. Lane M: DL marker; Lane 1: CA2-WT recombinant plasmid; Lane 2: enzyme digestion band of CA2-WT digested by MluⅠ, the length was 1028 bp (the arrow indicated). | + | <center>Fig. 2 Agarose Gel Electrophoresis of CA2-WT recombinant plasmid and its identification by enzyme digestion. Lane M: DL marker; Lane 1: CA2-WT recombinant plasmid; Lane 2: enzyme digestion band of CA2-WT plasmid digested by MluⅠ, the length was 1028 bp (the arrow indicated). |
</center> | </center> | ||
<h3>Induced expression of CA2-WT</h3> | <h3>Induced expression of CA2-WT</h3> | ||
| − | <p>The CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and the | + | <p>The CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and positive clones were screened by kanamycin resistance. Then, the recombinant E. coli BL21 (DE3) were propagated and CA2-WT expression was induced at the fourth hour of cell cultivation using an IPTG concentration of 500 μM. Cells were lysed by sonication on ice, and the obtained crude extract was centrifuged to separate supernatant and debris, and both fractions were subjected to SDS-PAGE and Western Blot (Fig. 3 and Fig. 4). The arrow indicated in Fig. 3 was the band of CA2 protein as the molecular weight of CA2 is about 30.6 kDa. It can be seen from lanes 1 and 2 that the CA2-WT expression was significantly induced with IPTG incubation. Results from lanes 3-6 indicated that the induced expression of CA2 mainly existed in soluble form in the cell lysate supernatant. The correctness of CA2 protein was also confirmed by Western blot assay in Fig. 4. In consequence, the results above demonstrate that an engineered E. coli BL21 (ED3) strain that expresses CA2-WT has been constructed. |
| + | </p> | ||
<div align="center"> | <div align="center"> | ||
https://static.igem.org/mediawiki/parts/c/cb/T--AHUT_China--_3part.jpg</div> | https://static.igem.org/mediawiki/parts/c/cb/T--AHUT_China--_3part.jpg</div> | ||
| − | <center>Fig. 3 SDS-PAGE analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain. | + | <center>Fig. 3 SDS-PAGE analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain. |
</center> | </center> | ||
<div align="center"> | <div align="center"> | ||
| − | https://static.igem.org/mediawiki/parts/ | + | |
| − | <center>Fig. | + | https://static.igem.org/mediawiki/parts/6/68/T--AHUT_China--_zuihou1245.jpg</div> |
| + | <center>Fig. 4 Western blot analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain. | ||
</center> | </center> | ||
| − | <h3>Purification of CA2-WT</h3> | + | <h3>Purification of CA2-WT protein</h3> |
| − | <p>After confirming that CA2 | + | <p>After confirming that CA2-WT could be expressed in E. coli BL21 (DE3), CA2-WT protein was further purified with nickel column, and the resulting protein had a molecular mass corresponding to CA2-WT protein (Fig. 5). Western blot analysis showed the protein to be recognized by antibodies specifically recognizing histidine-tag (Fig. 6). |
| + | </p> | ||
<div align="center"> | <div align="center"> | ||
https://static.igem.org/mediawiki/parts/0/0f/T--AHUT_China--_5part.jpg</div> | https://static.igem.org/mediawiki/parts/0/0f/T--AHUT_China--_5part.jpg</div> | ||
| − | <center>Fig. 5 CA2 was purified with Ni column; fractions were analyzed by SDS-PAGE. Lane M: Protein marker; Lane 1: Supernatant after cell lysate centrifugation; Lane 2: Flow through; Lane 3: Wash with 50mM Tris, 150mM NaCl, 20 mM Imidazole, pH 8.0; Lane 4: Elute with 50mM Tris, 150mM NaCl, 50 mM Imidazole, pH 8.0; Lane 5: Elute with 50mM Tris, 150mM NaCl, 500 mM Imidazole, pH 8.0.</center> | + | <center>Fig. 5 CA2 was purified with Ni column; fractions were analyzed by SDS-PAGE. Lane M: Protein marker; Lane 1: Supernatant after cell lysate centrifugation; Lane 2: Flow through; Lane 3: Wash with 50mM Tris, 150mM NaCl, 20 mM Imidazole, pH 8.0; Lane 4: Elute with 50mM Tris, 150mM NaCl, 50 mM Imidazole, pH 8.0; Lane 5: Elute with 50mM Tris, 150mM NaCl, 500 mM Imidazole, pH 8.0. |
| + | </center> | ||
<div align="center"> | <div align="center"> | ||
| − | + | https://static.igem.org/mediawiki/parts/4/4e/T--AHUT_China--_zhuhou1411.jpg</div> | |
| − | https://static.igem.org/mediawiki/parts/ | + | <center>Fig. 6 Western blot analysis of CA2-WT protein. Lane M: Protein marker; Lane 1: Purified CA2-WT. |
| − | <center>Fig. 6 | + | |
</center> | </center> | ||
Latest revision as of 02:50, 18 October 2018
Carbonic anhydrase 2
This part is the coding sequence (CDS) of wild-type human carbonic anhydrase 2 (CA2) with a His-tag placed at the C-terminal. CA2 is a metalloenzyme that can efficiently catalyze the formation of HCO3- and H+ from CO2 with high catalytic efficiency and fast reaction rate.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Construction of wild-type human carbonic anhydrase 2 (CA2-WT) expression plasmid
We first synthesized the sequence of CA2-WT, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig. 1 and Fig. 2).
Induced expression of CA2-WT
The CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and positive clones were screened by kanamycin resistance. Then, the recombinant E. coli BL21 (DE3) were propagated and CA2-WT expression was induced at the fourth hour of cell cultivation using an IPTG concentration of 500 μM. Cells were lysed by sonication on ice, and the obtained crude extract was centrifuged to separate supernatant and debris, and both fractions were subjected to SDS-PAGE and Western Blot (Fig. 3 and Fig. 4). The arrow indicated in Fig. 3 was the band of CA2 protein as the molecular weight of CA2 is about 30.6 kDa. It can be seen from lanes 1 and 2 that the CA2-WT expression was significantly induced with IPTG incubation. Results from lanes 3-6 indicated that the induced expression of CA2 mainly existed in soluble form in the cell lysate supernatant. The correctness of CA2 protein was also confirmed by Western blot assay in Fig. 4. In consequence, the results above demonstrate that an engineered E. coli BL21 (ED3) strain that expresses CA2-WT has been constructed.
Purification of CA2-WT protein
After confirming that CA2-WT could be expressed in E. coli BL21 (DE3), CA2-WT protein was further purified with nickel column, and the resulting protein had a molecular mass corresponding to CA2-WT protein (Fig. 5). Western blot analysis showed the protein to be recognized by antibodies specifically recognizing histidine-tag (Fig. 6).
