Difference between revisions of "Part:BBa K2547000:Experience"

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Applications of BBa_K2547000

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).

Fig. 1 Map of CA2-WT recombinant vector

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).

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, and the correctness of CA2 protein was also confirmed by Western blot assay in Fig. 4. It can be seen from lanes 1 and 2 in both Figures 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. In consequence, the results above demonstrate that an engineered E. coli BL21 (ED3) strain that expresses CA2-WT has been constructed.

Fig. 3 SDS-PAGE analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain.

Fig. 4 Western blot analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain.

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 and Fig. 6). Western blot analysis showed the protein to be recognized by antibodies specifically recognizing histidine-tag (Fig. 6).

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.

Fig. 6 SDS-PAGE and Western blot analysis of CA2-WT protein. Lane 1: Positive control (BSA); Lane 2: purified CA2-WT; Lane 3: Purified CA2-WT.

User Reviews

UNIQ3b19dcad23e8e8e5-partinfo-00000000-QINU UNIQ3b19dcad23e8e8e5-partinfo-00000001-QINU

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 ===Applications of BBa_K2547000===
-<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>
+<p>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).
+<br></p>
 <div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_par1t.jpg
@@ Line 15: / Line 17: @@
 <div align="center">
 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>
 <h3>Induced expression of CA2-WT</h3>
-<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>
+<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, and the correctness of CA2 protein was also confirmed by Western blot assay in Fig. 4. It can be seen from lanes 1 and 2 in both Figures 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. 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">&nbsp;&nbsp;&nbsp;&nbsp;
 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>
 <div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 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>Fig. 4 Western blot analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain.
 </center>
-<h3>Purification of CA2-WT</h3>
+<h3>Purification of CA2-WT protein</h3>
-<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>
+<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 and Fig. 6). Western blot analysis showed the protein to be recognized by antibodies specifically recognizing histidine-tag (Fig. 6).
+</p>
 <div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 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">&nbsp;&nbsp;&nbsp;&nbsp;
 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>Fig. 6 SDS-PAGE and Western blot analysis of CA2-WT protein. Lane 1: Positive control (BSA); Lane 2: purified CA2-WT; Lane 3: Purified CA2-WT.
 </center>