Part:BBa_K2232000

TSLV1-CA

This part is the coding sequence (CDS) of Carbonic anhydrase (CA) from The polyextremophilic bacterium Bacillus halodurans TSLV1 (MTCC 10961, 16S rDNA Acc. No. HQ235051).CA is a metalloenzyme with zinc, which is highly efficient and one of the fastest enzymes catalyzes the reversible hydration of CO2 forming bicarbonate and protons rapidly.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 540
  Illegal NotI site found at 827
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 520
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 65
  Illegal SapI.rc site found at 223
  Illegal SapI.rc site found at 535
  

iGEM2018 AHUT-China

<--User Reviews-->
    Yuru_chen
    We sequenced the existing part Carbonic anhydrase (csoS3) of the carboxysome of Halothiobacillus neapolitanus (BBa_K1465205) to generate a new PART (BBa_K2547003 (Carbonic anhydrase (csoS3)-His-Tag) (Fig. 1)

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

    Specifically, the coding sequence of Carbonic anhydrase csoS3 was codon-optimized, and His-tag was added to the end, so that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and had good carbonic anhydrase activity.
    First, we synthesized the original coding sequence of csoS3 and the coding sequence after codon optimization, and cloned into the expression vector pET-30a(+) respectively. The correctness of the two plasmids was verified by PCR (Fig. 2).

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

    Subsequently, we examined the expression of two csoS3 in E. coli. The results are shown in Figure 3. The expression of the codon-optimized plasmid in E. coli is very low, and the codon-optimized csoS3 is in E. coli. The expression increased significantly.

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Fig. 3 SDS-PAGE analysis of Carbonic anhydrase csoS3-His-Tag 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.

    On this basis, we further purified E. coli expressing new part csoS3 to obtain purified csoS3 carbonic anhydrase (Fig. 4), and carried out enzyme activity assay by esterase method. The enzyme activity assay showed that csoS3 had certain The enzyme activity was 22.84 U/mL.

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Fig. 4 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.

iGEM2017 SZU-China

To realize the self-healing of cracks in concrete, we need to increase the mineralization capacity of B.subtilis. The Healer in our project is Carbonic anhydrase(CA) , which catalyzes the hydration of CO₂ to produce HCO₃^- and captures free Ca²⁺ with OH^- in the environment to form Calcium carbonate precipitation. The new part TSLV1-CA (BBa_K2232014) expresses and functiones intracellularly. We constructed a shuttle vector to transform this part and the positive clones was confirmed by nucleic acid electrophoresis(Fig.1).

Fig.1 1% Agarose Gel Electrophoresis of Vector_ TSLV1-CA 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 TSLV1-CA was 949 bp and the blank vector was 6785 bp.

The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining(Fig.2).

Fig.2 SDS-PAGE analysis of endocellular protein of original B.subtilis and the transformant of CA. Lane M: Marker ladder; Lane 1: Modified strain WB800_ TSLV1-CA; Lane 2: Modified strain WB800_ OF4-CA; Lane 3: Original strain WB800. Lane 1 and lane 2 have a band of 35~37kd respectively (in red box), which correspond with molecular weight of TSLV1-CA (35kDa) and OF4-CA (34.5kDa).

For determining the activity of CA, hydration of CO₂ was measured using electrometric Wilbur–Anderson assay according to Khalifah et al. (1991) with certain modifications. The assay was performed at 4 °C by adding 0.5 mL of the crude enzyme solution (0.5 ml distilled water in blank group) to 10 mL of 30mM PBS (pH 8.0). The reaction was initiated by adding 5.0 mL of ice-cold CO₂ saturated water. The time interval for the pH to drop by 1.5 unit (from 8.0 to 6.5) due to protons released during hydration of CO2 was measured. The reactions were performed in triplicates and average of three replicates was used in calculations. We calculated the activity according to the formula U= (T0 –T1)/ T0, where T0 and T1 represent time for pH change of blank group and samples group respectively. The CA activity was shown in Fig.3.

Fig.3 CA activity of crude enzyme solution from measured by Brownell’s method