Difference between revisions of "Part:BBa K2547003"
 
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<partinfo>BBa_K2547003 short</partinfo>
 
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The carbonic anhydrase from Halothiobacillus neapolitanus converts the incoming bicarbonate into carbon dioxide in the carboxyl group, a step that is essential for CO 2 fixation. We have codon-optimized the coding sequence (CDS) to make the carbonic anhydrase gene more suitable for expression in E. coli. Simultaneously linking a histidine tag (His-Tag) to the coding sequence (CDS) is beneficial to carbonic anhydride.  
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This is an improved part of carbonic anhydrase (csoS3) from Halothiobacillus neapolitanus which converts the incoming bicarbonate into carbon dioxide in the carboxysome, a step that is essential for CO2 fixation. We have optimized codon sequence in the coding sequence (CDS) to make the carbonic anhydrase gene more suitable for expression in E. coli. In addition to codon optimization, we have added a His-tag to the C-terminal of the CDS that is used for purification of carbonic anhydrase protein.
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===Usage and Biology===
 
===Usage and Biology===
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<partinfo>BBa_K2547003 parameters</partinfo>
 
<partinfo>BBa_K2547003 parameters</partinfo>
 
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&nbsp;&nbsp;&nbsp;&nbsp;The coding sequence of Carbonic anhydrase csoS3 from original part was codon-optimized, and also a His tag was added to the end, to ensure that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and retained potent carbonic anhydrase activity (Fig. 1).<br></p>
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<p>The coding sequence of Carbonic anhydrase csoS3 from original part was codon-optimized, and also a His tag was added to the end, to ensure that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and retained potent carbonic anhydrase activity (Fig. 1).<br></p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;https://static.igem.org/mediawiki/parts/1/14/T--AHUT_China--_pa1rt.jpg
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;https://static.igem.org/mediawiki/parts/1/14/T--AHUT_China--_pa1rt.jpg
 
</div>
 
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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: 14px;text-align: center;">Fig. 1 Map of Carbonic anhydrase csoS3-His expression vector
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<center>Fig. 1 Map of Carbonic anhydrase csoS3-His expression vector
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<p>First, the original coding sequence of csoS3 and the coding sequence with codon optimization were synthesized, and cloned into the pET-30a (+) expression vectors, respectively. The correctness of the two recombinant plasmids was verified by PCR (Fig. 2).</p>     
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&nbsp;&nbsp;&nbsp;&nbsp;First, the original coding sequence of csoS3 and the coding sequence with codon optimization were synthesized, and cloned into the pET-30a (+) expression vectors, respectively. The correctness of the two recombinant plasmids was verified by PCR (Fig. 2).</p>     
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<div align="center">https://static.igem.org/mediawiki/parts/d/d6/T--AHUT_China--_p2art.jpg
 
<div align="center">https://static.igem.org/mediawiki/parts/d/d6/T--AHUT_China--_p2art.jpg
 
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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: 14px;text-align: center;">Fig. 2 Agarose Gel Electrophoresis of Carbonic anhydrase csoS3 expression vectors and its identification by PCR. Lane M: DL marker; Lane 1: expression vector of csoS3 new part; Lane 2: PCR band of expression vector of csoS3 new part, the length was 1620 bp; Lane 3: expression vector of csoS3 original part; Lane 4: PCR band of expression vector of csoS3 original part, the length was 1620 bp.
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<center>Fig. 2 Agarose Gel Electrophoresis of Carbonic anhydrase csoS3 expression vectors and its identification by PCR. Lane M: DL marker; Lane 1: expression vector of csoS3 new part; Lane 2: PCR band of expression vector of csoS3 new part, the length was 1620 bp; Lane 3: expression vector of csoS3 original part; Lane 4: PCR band of expression vector of csoS3 original part, the length was 1620 bp.
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Subsequently, the expression of two csoS3 plasmids in E. coli was detected via SDS-PAGE and Coomassie blue staining. As shown in Fig. 3, the result presented that the expression of csoS3 original part in E. coli was relatively low, and the expression of codon-optimized csoS3 new part in E. coli was higher than original part.</p>
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<p>Subsequently, the expression of two csoS3 plasmids in E. coli was detected via SDS-PAGE and Coomassie blue staining. As shown in Fig. 3, the result presented that the expression of csoS3 original part in E. coli was relatively low, and the expression of codon-optimized csoS3 new part in E. coli was higher than original part.</p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
https://static.igem.org/mediawiki/parts/0/08/T--AHUT_China--_p3art.jpg</div>
 
https://static.igem.org/mediawiki/parts/0/08/T--AHUT_China--_p3art.jpg</div>
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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: 14px;text-align: center;">Fig. 3 SDS-PAGE and Coomassie blue staining of Carbonic anhydrase csoS3 plasmids expressed in E. coli BL21(DE3) strains. The arrow indicated was the bands of csoS3. Lane 1: Negative control (cell lysate without IPTG induction) of new part; Lane 2: Cell lysate with induction for 6 h at 37 ℃ of new part; Lane 3: Negative control (cell lysate without IPTG induction) of original part; Lane 4: Cell lysate with induction for 6 h at 37 ℃ of original part.
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<center>Fig. 3 SDS-PAGE and Coomassie blue staining of Carbonic anhydrase csoS3 plasmids expressed in E. coli BL21(DE3) strains. The arrow indicated was the bands of csoS3. Lane 1: Negative control (cell lysate without IPTG induction) of new part; Lane 2: Cell lysate with induction for 6 h at 37 ℃ of new part; Lane 3: Negative control (cell lysate without IPTG induction) of original part; Lane 4: Cell lysate with induction for 6 h at 37 ℃ of original part.
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<p>&nbsp;&nbsp;&nbsp;&nbsp;To further demonstrate the activity of our new part, new part of csoS3 carbonic anhydrase was purified through Ni-chelating affinity chromatography and detected by SDS-PAGE and Coomassie blue staining, as shown in Fig. 4. Then, the activity of csoS3 was measured via esterase method, and the enzyme activity was about 22.84 U/mL.</p>
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<p>To further demonstrate the activity of our new part, new part of csoS3 carbonic anhydrase was purified through Ni-chelating affinity chromatography and detected by SDS-PAGE and Coomassie blue staining, as shown in Fig. 4. Then, the activity of csoS3 was measured via esterase method, and the enzyme activity was about 22.84 U/mL.</p>
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
<div align="center">&nbsp;&nbsp;&nbsp;&nbsp;
 
https://static.igem.org/mediawiki/parts/4/4b/T--AHUT_China--_p4art.jpg</div>
 
https://static.igem.org/mediawiki/parts/4/4b/T--AHUT_China--_p4art.jpg</div>
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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: 14px;text-align: center;">Fig. 4 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.
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<center>Fig. 4 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.
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<p>In conclusion, our results demonstrated that the function of csoS3 new part has been improved with higher expression than original part and activity retained.
 
</p>
 
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<p>&nbsp;&nbsp;&nbsp;&nbsp;In conclusion, our results demonstrated that the function of csoS3 new part has been improved with higher expression and activity than original part.</p>
 

Latest revision as of 14:42, 17 October 2018


Carbonic anhydrase (csoS3)-His

This is an improved part of carbonic anhydrase (csoS3) from Halothiobacillus neapolitanus which converts the incoming bicarbonate into carbon dioxide in the carboxysome, a step that is essential for CO2 fixation. We have optimized codon sequence in the coding sequence (CDS) to make the carbonic anhydrase gene more suitable for expression in E. coli. In addition to codon optimization, we have added a His-tag to the C-terminal of the CDS that is used for purification of carbonic anhydrase protein.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


The coding sequence of Carbonic anhydrase csoS3 from original part was codon-optimized, and also a His tag was added to the end, to ensure that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and retained potent carbonic anhydrase activity (Fig. 1).

    T--AHUT_China--_pa1rt.jpg
Fig. 1 Map of Carbonic anhydrase csoS3-His expression vector

First, the original coding sequence of csoS3 and the coding sequence with codon optimization were synthesized, and cloned into the pET-30a (+) expression vectors, respectively. The correctness of the two recombinant plasmids was verified by PCR (Fig. 2).

T--AHUT_China--_p2art.jpg
Fig. 2 Agarose Gel Electrophoresis of Carbonic anhydrase csoS3 expression vectors and its identification by PCR. Lane M: DL marker; Lane 1: expression vector of csoS3 new part; Lane 2: PCR band of expression vector of csoS3 new part, the length was 1620 bp; Lane 3: expression vector of csoS3 original part; Lane 4: PCR band of expression vector of csoS3 original part, the length was 1620 bp.

Subsequently, the expression of two csoS3 plasmids in E. coli was detected via SDS-PAGE and Coomassie blue staining. As shown in Fig. 3, the result presented that the expression of csoS3 original part in E. coli was relatively low, and the expression of codon-optimized csoS3 new part in E. coli was higher than original part.

     T--AHUT_China--_p3art.jpg
Fig. 3 SDS-PAGE and Coomassie blue staining of Carbonic anhydrase csoS3 plasmids expressed in E. coli BL21(DE3) strains. The arrow indicated was the bands of csoS3. Lane 1: Negative control (cell lysate without IPTG induction) of new part; Lane 2: Cell lysate with induction for 6 h at 37 ℃ of new part; Lane 3: Negative control (cell lysate without IPTG induction) of original part; Lane 4: Cell lysate with induction for 6 h at 37 ℃ of original part.

To further demonstrate the activity of our new part, new part of csoS3 carbonic anhydrase was purified through Ni-chelating affinity chromatography and detected by SDS-PAGE and Coomassie blue staining, as shown in Fig. 4. Then, the activity of csoS3 was measured via esterase method, and the enzyme activity was about 22.84 U/mL.

     T--AHUT_China--_p4art.jpg
Fig. 4 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.

In conclusion, our results demonstrated that the function of csoS3 new part has been improved with higher expression than original part and activity retained.