Difference between revisions of "Team:AHUT China/New Parts"

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         <p style="text-align: center; font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 36px;"><strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400; color: #000000;">Experimental results</strong></p>
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         <p style="text-align: center; font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 36px;"><strong style="font-family: Segoe, 'Segoe UI', 'DejaVu Sans', 'Trebuchet MS', Verdana, sans-serif; font-style: normal; font-weight: 400; color: #000000;">Parts results</strong></p>
 
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                   <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px; "><span style="font-size: 24px">The </span>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>                         
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                   <p style="font-family: 'Arial Unicode MS', 'Microsoft YaHei UI', 'Microsoft YaHei UI Light', '华文细黑', '微软雅黑', '幼圆'; font-size: 18px; "><span style="font-size: 24px">The </span>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>                         
 
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Revision as of 00:34, 18 October 2018

Royal Hotel

Royal Hotel

Parts results




BBa_K2547000: Construction of CA2-WT expression plasmid

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).




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 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. 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.




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




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








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.

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 Western blot analysis of CA2-WT protein. Lane M: Protein marker; Lane 1: Purified CA2-WT.








BBa_K2547004 : Construction of CA2（L203K）expression plasmid

We first synthesized the sequence of the mutant 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).




Fig. 1 Map of CA2 (L203K) recombinant vector




Fig. 2 Agarose Gel Electrophoresis of CA2(L203K) recombinant plasmid and its identification by enzyme digestion （NdeⅠand Hind Ⅲ）. Lane M: DNA marker; Lane 1: CA2(L203K) recombinant plasmid; Lane 2: enzyme digestion band of CA2(L203K) , the length was 825 bp (the arrow indicated).







Induced expression of CA2(L203K)

The CA2(L203K) expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 (L203K) expression, and the bacterial solution was sonicated, followed by SDS-PAGE(figure 3), the size of CA2(L203K) is known to be 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2(L203K) band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significant to the expression of CA2 which was induced, and it can be seen from lanes 3-6 that the induced expression of CA2 was mainly expressed in soluble form in the supernatant of the bacterial liquid. The above results indicated that we successfully obtained E. coli expressing CA2(L203K).




Fig. 3 SDS-PAGE analysis for CA2(L203K) cloned in pET-30a(+) and expressed in BL21(DE3) strain.









Purification of CA2(L203K) protein

After confirming that CA2(L203K) can be induced by E. coli BL21(DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2(L203K) protein. Figure 4 shows the results. We have obtained a highly purified mutant CA2 protein.




Fig. 4 SDS-PAGE and Western blot analysis of CA2(L203K). Lane 1: Negative control; Lane 2: purified CA2(L203K) protein










Determination of protease activity of CA2 and CA2 (L203K)

We determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As shown in Figure 5 and Figure 6, mutant CA2 (L203K) has higher enzymatic activity than wild-type CA2.




Fig. 5 Colorimetric assay of CA2 activity




Fig. 6 Esterase activity analysis of CA2 protein









Analysis of Thermal Stability of CA2 and CA2 (L203K)

We examined the activity of carbonic anhydrase in wild-type and mutant CA2 at different times and temperatures by esterase method. The results are shown in Figure 7. As the temperature increases, especially at 55 ° C and 65 ° C, The enzymatic activity of wild type CA2 was significantly decreased, while the mutant CA2 still had higher activity, indicating that CA2 (L203K) has better thermal stability.




Fig. 7 Activity of purified CA2-WT and CA2 (L203K) under indicated temperatures and time points.