Part:BBa_K2232000:Experience

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Contribution from iGEM2022 ZJU-China

Group: ZJU-China 2022
Author: Junyi Liu, Jiachen Li
Summary: In 2022, ZJU-China used this part in their project, experimentally characterized the function of the carbonic anhydrase encoded by this part.We also performed codon optimization and compared the function of the parts before and after optimization.

Characterization

We've created Part:BBa_K4202004 ,which is improved by codon optimization from Part:BBa_K2232000. Then we have done a series of control experiments to compare the function of two parts. Both of the two parts encodes carbonic anhydrase, so we name the carbonic anhydrase expressed by BBa_K2232000 as CA1, while name the carbonic anhydrase expressed by BBa_K4202004 as CA2.

Expression of BBa_K2232000

After chemical transformation of plasmid with this part to Basillus subtilis WB600, the transformed Bacillus subtilis WB600 were cultured in optimized LB and SMM medium, and obtained crude enzyme solution by centrifugation and ultrasonic disruption.Then we detected the molecular mass by SDS-PAGE and coomassie blue staining.
SDS-PAGE displayed bands of 37kDa and 74kDa for CA monomer and dimer, which didn' t exist in the control group(Fig.1-1).

Fig 1-1 SDS-PAGE and Coomassie brilliant blue staining results of whole protein lysates of strain CA1, strain CA2 and blank WB600. Lane 1: Protein Ladder; Lane2: CA2 strain grown in LB medium, Lane3: CA2 strain grown in SMM medium, Lane4: CA1 strain grown in LB medium, Lane5: CA1 strain grown in SMM medium, Lane6: blank WB600 strain.

Determination the ability of TSLV-BS-CA to catalyze the hydration of CO₂

To measure the activity of CA, we used the modified Wilbur-Anderson's method. As we know, CA can catalyze CO₂ hydration and at the same time release H⁺ reducing the pH. According to that, we chose bromothymol blue, an acid-base indicator that appears yellow when pH≤6 and blue when pH > 7.6. Therefore, the color development of bromothymol blue can indirectly reflect the change of pH from 8.0 to 6.0 by CA.

After adding ice-saturated CO₂ solution for 10min, the color of the tubes containing crude enzyme solution CA1 and CA2 began to change, indicating that the pH of the solution began to decrease. After 10min, the tubes containing the crude enzyme solution showed significant discoloration, and after 5 days, the tubes containing the CA1 crude enzyme solution turned completely yellow, implying that the pH had decreased from 8.0 to 6.0 due to the formation of H⁺ during CO₂ hydration(Fig.1-3).

The activity of CA1 and CA2 were verified in this experiment, but the enzyme activities were weak, possibly due to low enzyme expression or insufficient concentration of unpurified enzyme. In addition, the catalytic rate of CA2 crude enzyme solution was lower than that of CA1.

Fig 1-3 The activity of CA detected by the Wilbur-Anderson's method. From left to right, the four tubes were pH=6.0 Tris-HCl buffer with bromothymol blue indicator, reaction system with blank WB600 lysate, CA2 crude enzyme solution, CA1 crude enzyme solution. A: Initial reaction solution（0min); B: 10min after adding ice-saturated CO₂ solution; C: 5d after adding ice-saturated CO₂ solution.

Determination the ability of TSLV-BS-CA to catalyze the precipitation of CaCO₃

To test the ability of engineered Bacillus subtilis WB600 to precipitate CaCO₃, we cultured the engineered bacteria in 30ml of LB medium at 25℃ for 3 days and added 5 ml of 100mM CaCl₂ solution on the first and second days. We filtered the culture medium through a Whatman membrane filter paper to separate the bacteria and CaCO₃. The bacteria and CaCO₃ were dried and weighed, respectively. We can calculate CaCO₃ precipitation capacity of engineered bacteria by the formula:CaCO₃ dry weight (mg)/cell dry weight (g).

After three days of cultivation, we could clearly see that the culture medium of the CA1 and CA2 transformant became turbid(Fig.1-4 A). The precipitate was filtered and dried.(Fig.1-4B). According to the the foemula:CaCO₃ production capacity = CaCO₃ dry weight (mg)/cell dry weight (g), we found that the the precipitation efficiency of CA1 was higher than that of CA2(Fig.1-4C).

Fig 1-4 A: The turbidity of the culture solution. B: Filtered and dried sediment products. C: CaCO₃ productions by CA1 and CA2 for 3d.

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Yuru_chen
In 2018, AHUT_China iGEM team has characterized the output of this part in a novel chassis E. coli BL21(DE3). The result was documented in the experience page and the main page of (BBa_K2232000).

Contribution from iGEM2018 AHUT_China

<p>The sequence of (BBa_K2232000) was synthesized and cloned into the expression plasmid pET-30a(+) to obtain the recombinant expression vector (Fig. 1).

Fig. 1 Agarose Gel Electrophoresis of TSLV1-CA recombinant plasmid and its identification by PCR. Lane M: DL marker; Lane 1: TSLV1-CA recombinant plasmid; Lane 2: PCR band of TSLV1-CA, the length was 894 bp.

Then, the TSLV1-CA expression plasmid was transformed into E. coli BL21 (DE3) strain, and positive clones were screened by kanamycin resistance. The positive clones were further propagated and induced with IPTG (isopropyl thiogalactoside), followed by protein extraction from lysates of bacterial solution. The expression of TSLV1-CA was identified by Western blot analysis. The results are shown in Fig. 2, indicating that the coding sequence of (BBa_K2232000) can be expressed in our chassis E. coli BL21 (DE3).

Fig. 2 Western blot analysis of protein extracted from lysates of TSLV1-CA expressed E.coli BL21(DE3) strain

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

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