Coding

Part:BBa_K2232000

Designed by: Wenkai Hu   Group: iGEM17_SZU-China   (2017-10-17)
Revision as of 10:17, 11 October 2018 by YuruChen (Talk | contribs)


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


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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