Difference between revisions of "Part:BBa K2547004:Experience"
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===Applications of BBa_K2547004===
 
===Applications of BBa_K2547004===
  
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<p>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).<br></p>
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<h3>Construction of mutant human carbonic anhydrase 2 (CA2 (L203K)) expression plasmid
 +
</h3>
 +
<p>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).
 +
<br></p>
 +
<p>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.
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</p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_par1t.jpg
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_par1t.jpg
 
</div>
 
</div>
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<div align="center">https://static.igem.org/mediawiki/parts/e/ee/T--AHUT_China--_par2t.jpg</div>
 
<div align="center">https://static.igem.org/mediawiki/parts/e/ee/T--AHUT_China--_par2t.jpg</div>
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<center>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).
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<center>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).
 
</center>
 
</center>
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<h3>Induced expression of CA2(L203K)</h3>
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<h3>Induced expression of CA2 (L203K) protein
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<p>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). </p>
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</h3>
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<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;https://static.igem.org/mediawiki/parts/6/6d/T--AHUT_China--_par3t.jpg</div>
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<p>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).  
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<center>Fig. 3 SDS-PAGE analysis for CA2(L203K) cloned in pET-30a(+) and expressed in BL21(DE3) strain.
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</p>
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<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;https://static.igem.org/mediawiki/parts/d/d6/T--AHUT_China--78947578.jpg</div>
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<center>Fig. 3 SDS-PAGE analysis for CA2 (L203K) cloned in pET-30a(+) vector and expressed in BL21(DE3) strain.
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</center>
 
</center>
  
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<h3>Purification of CA2(L203K) protein</h3>
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<h3>Purification of CA2 (L203K) protein
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<p>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.</p>
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</h3>
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<p>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).
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</p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
https://static.igem.org/mediawiki/parts/1/1d/T--AHUT_China--_par9t.jpg</div>
 
https://static.igem.org/mediawiki/parts/1/1d/T--AHUT_China--_par9t.jpg</div>
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<center>Fig. 4 SDS-PAGE and Western blot analysis of CA2(L203K). Lane 1: Negative control; Lane 2: purified CA2(L203K) protein
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<center>Fig. 4 SDS-PAGE and Western blot analysis of CA2 (L203K) protein. Lane 1: Negative control; Lane 2: purified CA2 (L203K) protein.
 
</center>
 
</center>
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<h3>Determination of protease activity of CA2 and CA2 (L203K)</h3>
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<h3>Enzyme activity assay of CA2-WT and CA2 (L203K) protein
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<p>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.</p>
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</h3>
 +
<p>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).
 +
</p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
https://static.igem.org/mediawiki/parts/6/68/T--AHUT_China--_par5t.jpg</div>
 
https://static.igem.org/mediawiki/parts/6/68/T--AHUT_China--_par5t.jpg</div>
 
<center>Fig. 5 Colorimetric assay of CA2 activity
 
<center>Fig. 5 Colorimetric assay of CA2 activity
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</center>
 
</center>
 
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https://static.igem.org/mediawiki/parts/8/86/T--AHUT_China--_par6t.jpg</div>
 
https://static.igem.org/mediawiki/parts/8/86/T--AHUT_China--_par6t.jpg</div>
 
<center>Fig. 6 Esterase activity analysis of CA2 protein
 
<center>Fig. 6 Esterase activity analysis of CA2 protein
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</center>
 
</center>
 
<h3>
 
<h3>
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Analysis of Thermal Stability of CA2 and CA2 (L203K)</h3>
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Thermal stability studies of CA2-WT and CA2 (L203K) protein
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<p>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.</p>
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</h3>
 +
<p>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.
 +
</p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
  
 
https://static.igem.org/mediawiki/parts/f/fc/T--AHUT_China--_par7t.jpg</div>
 
https://static.igem.org/mediawiki/parts/f/fc/T--AHUT_China--_par7t.jpg</div>
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<center>Fig. 7 Activity of purified CA2-WT and CA2 (L203K) under indicated temperatures and time points.
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<center>Fig. 7 Activity of purified CA2-WT and CA2 (L203K) protein under indicated temperatures and time points.
 
</center>
 
</center>
  

Revision as of 14:08, 15 October 2018


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Applications of 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.

    T--AHUT_China--_par1t.jpg
Fig. 1 Map of CA2 (L203K) recombinant vector
T--AHUT_China--_par2t.jpg
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).

    T--AHUT_China--78947578.jpg
Fig. 3 SDS-PAGE analysis for CA2 (L203K) cloned in pET-30a(+) vector 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).

     T--AHUT_China--_par9t.jpg
Fig. 4 SDS-PAGE and Western blot analysis of CA2 (L203K) protein. 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).

     T--AHUT_China--_par5t.jpg
Fig. 5 Colorimetric assay of CA2 activity
     T--AHUT_China--_par6t.jpg
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.

     T--AHUT_China--_par7t.jpg
Fig. 7 Activity of purified CA2-WT and CA2 (L203K) protein under indicated temperatures and time points.

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