Part:BBa_K2232000:Experience

iGEM2018 AHUT_China

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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.
    The sequence of BBa_K2232000 was synthesized and cloned it 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).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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