Difference between revisions of "Part:BBa K2547000:Experience"
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===Applications of BBa_K2547000=== | ===Applications of BBa_K2547000=== | ||
| − | <p | + | <p>We first synthesized the sequence of human carbonic anhydrase CA2, 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/7/7f/T--AHUT_China--_par1t.jpg | https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_par1t.jpg | ||
</div><br><br> | </div><br><br> | ||
| − | < | + | <center>Fig. 1 Map of CA2-WT recombinant vector |
| − | </ | + | </center> |
<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> |
<h3>Induced expression of CA2-WT</h3> | <h3>Induced expression of CA2-WT</h3> | ||
| − | <p | + | <p>The CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 expression, and the bacterial solution was sonicated, followed by SDS-PAGE and Western Blot(Fig. 3 and Fig. 4) .It shows that the size of CA2 is 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2 band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significantly induced. The expression of CA2, and it can be seen from lanes 3-6, that the induced expression of CA2 is mainly expressed in a soluble form in the supernatant of the bacterial liquid.The above results indicate that we successfully obtained E. coli which expresses CA2.</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> |
<div align="center"> | <div align="center"> | ||
https://static.igem.org/mediawiki/parts/0/0e/T--AHUT_China--_4part.jpg</div> | https://static.igem.org/mediawiki/parts/0/0e/T--AHUT_China--_4part.jpg</div> | ||
| − | < | + | <center>Fig. 4Western blot analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain. |
| − | </ | + | </center> |
<h3>Purification of CA2-WT</h3> | <h3>Purification of CA2-WT</h3> | ||
| − | <p | + | <p>After confirming that CA2 can be induced by E. coli BL21 (DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2 protein. The results in the figure (Figures 5 and 6) illustrate that we have obtained a higher purity CA2 protein.</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> |
<div align="center"> | <div align="center"> | ||
https://static.igem.org/mediawiki/parts/8/88/T--AHUT_China--_125part.jpg</div> | https://static.igem.org/mediawiki/parts/8/88/T--AHUT_China--_125part.jpg</div> | ||
| − | < | + | <center>Fig. 6 SDS-PAGE and Western blot analysis of CA2. Lane 1: Positive control (BSA); Lane 2: purified CA2; Lane 3: Purified CA2. |
| − | </ | + | </center> |
| + | |||
===User Reviews=== | ===User Reviews=== | ||
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Revision as of 12:36, 13 October 2018
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Applications of BBa_K2547000
We first synthesized the sequence of human carbonic anhydrase CA2, 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 the cultured liquid was subjected to IPTG-induced CA2 expression, and the bacterial solution was sonicated, followed by SDS-PAGE and Western Blot(Fig. 3 and Fig. 4) .It shows that the size of CA2 is 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2 band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significantly induced. The expression of CA2, and it can be seen from lanes 3-6, that the induced expression of CA2 is mainly expressed in a soluble form in the supernatant of the bacterial liquid.The above results indicate that we successfully obtained E. coli which expresses CA2.
Purification of CA2-WT
After confirming that CA2 can be induced by E. coli BL21 (DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2 protein. The results in the figure (Figures 5 and 6) illustrate that we have obtained a higher purity CA2 protein.
User Reviews
UNIQ33d31989d232e4ba-partinfo-00000000-QINU UNIQ33d31989d232e4ba-partinfo-00000001-QINU