Part:BBa_K1465205

Carbonic anhydrase (csoS3) of the carboxysome of Halothiobacillus neapolitanus

The carbonic anhydrase from Halothiobacillus neapolitanus converts incoming hydrogen carbonate into carbon dioxide inside the carboxysome. This step is essential for the CO₂ fixation.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 166
  
• 1000
  COMPATIBLE WITH RFC[1000]

User Reviews

Yuru_chen
In 2018, AHUT_China iGEM team has changed the coding sequence (CDS) of the original part (BBa_K1465205) with codon-optimization and a histidine tag (His-Tag) added, forming a new Part (BBa_K2547003).

Contribution from iGEM2018 AHUT_China

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).

Fig. 1 Map of Carbonic anhydrase csoS3-His-Tag expression vector <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).

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.

    

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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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.

    

<center>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 and activity than original part.

User Reviews

james_zhang

In 2017 SZU-China iGEM team changed the sequence of original part(BBa_K1465205) and the new part BBa_K2232022 was submited.

Contribution from iGEM2017 SZU-China

We add the RBS of Bacillus subtilis and a segment of signal peptide at the upstream of cds of Carboxysomal Carbonic Anhydrase(csoS3) from Halothiobacillus neapolitanus, in order to achieve the expression and secretion of this part in the B.subtilis WB800. The construction of the shuttle expression-secretion vector_CsoS3 is shown as follow in figure 1.

Fig.1 Construction of the shuttle expression-secretion vector_CsoS3.

The new part (BBa_K2232022) was constructed into shuttle expression-secretion vector pP43NMK, and 1% Agarose Gel Electrophoresis of the plasmid verified the correct construction in figure 2.

Fig.2 1% Agarose Gel Electrophoresis of Vector_ CsoS3 and its identification by restriction digestion. Lane 1: Complete plasmid; Lane 2: Plasmid digested by KpnI and HindIII; Lane M: DL marker.The length of part CsoS3 was 1774 bp and the blank vector was 6785 bp.

The crude enzyme solution was obtained from the supernatant of fermentation broth of the Bacillus subtilis including the original strain WB800 and the recombinant WB800_ CsoS3. Since the fact that CA can also catalyze the hydration reaction of ester, which can be used to assay the activity of CA, we conducted measurement on CsoS3 according to a method from Verpoorte JA(1967), and the result(Fig.3) certified that the CsoS3 has high esterase activity.

Fig.3 The esterase activity of crude enzyme solution from the supernatant of fermentation broth of the Bacillus subtilis including the original strain WB800 and the recombinant WB800_ CsoS3.