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

Revision as of 03:23, 18 October 2018

Royal Hotel

Parts results

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 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 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).

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 mutant human carbonic anhydrase 2 (CA2 (L203K)) expression plasmid

Because wild-type CA2 has the fastest reaction rate at 37 °C and loses its activity at 50 °C, so it may be not suitable for using wide type CA2 to capture CO2 under industrial operating conditions. Therefore, we use molecular simulation to design new high-efficiency and stable carbonic anhydrases by improving their catalytic properties and stability. Basing on the simulation results above, we finally determined that the suitable mutation site of CA2 with high and stable activity was L203K (the 203th leucine mutated into lysine).
Therefore, we constructed an expression vector containing CA2 (L203K) coding sequence for following activity assay (Fig. 1). The obtained recombinant vector was verified by restriction enzyme digestion (Fig. 2) and sequencing.

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) protein

The CA2 (L203K) expression plasmid was transformed into E. coli BL21 (DE3), and its expression was induced with IPTG, and identified by SDS-PAGE analysis. The results showed that CA2 (L203K) could be expressed in BL21 (DE3) strain and existed in soluble form in the cell lysate supernatant (Fig. 3).

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

Purification of CA2(L203K) protein

In order to detect the enzyme activity of CA2 (L203K) protein, we further purify the crude protein extract by nickel column to obtain purified CA2 (L203K) protein. CA2 (L203K) was purified with high purity as indicated by a significant single protein band after SDS-PAGE and Western blot (Fig. 4).

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

Enzyme activity assay of CA2-WT and CA2 (L203K) protein

Next, we determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As indicated in Fig. 5, specific activity of mutant CA2 was about 2 times greater than that of wild-type enzyme. The kinetic constants (Km and Vmax) were calculated for esterase activity assay, and the result showed that CA2 (L203K) protein has a higher activity than CA2-WT (Fig. 6).

Fig. 5 Colorimetric assay of CA2 activity

Fig. 6 Esterase activity analysis of CA2 protein

Thermal stability studies of CA2-WT and CA2 (L203K) protein

We then investigated the effect of temperature on CA2 activity by esterase activity assay. As shown in Fig. 7, as the temperature increases, especially at 55 °C and 65 °C, the enzymatic activity of CA2-WT was significantly decreased, while the mutant CA2 still retain relatively high activity, indicating that CA2 (L203K) was more stable at high temperature and retained its activity.

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

- TEAM
- Team Members
- Attribution
- PROJECT
- Overview
- Background
- Protocol
- Design
- Demonstrate
- Interlab
- Result
- Notebook
- Parts
- Improved Parts
- New Parts
- Modeling
- Model
- Collaboration
- Collaboration

Anhui University of Technology is located in the national civilized city, Nanjing metropolitan core cities - Ma'anshan City, Anhui Province. Anhui University of Technology is an engineering, metallurgical features, engineering, economics, management, arts, science, law and other disciplines coordinated development of key construction in Anhui multidisciplinary university, with "The Master, Bo" complete the multi-level education training system.

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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>
 <br><br><br>
-                     <div align="center"><img src="https://static.igem.org/mediawiki/2018/f/ff/T--AHUT_China--10121.3.png" width="600"  alt=""/></div>
+                     <div align="center"><img src="https://static.igem.org/mediawiki/2018/9/94/T--AHUT_China--_dskhcdkv.jpg" width="600"  alt=""/></div>
 <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: 14px; text-align:center;">Fig. 3 SDS-PAGE analysis for CA2 cloned in pET-30a(+) and expressed in BL21(DE3) strain.</p>
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