Part:BBa_K2232014

P43_TSLV1-CA-alpha_rrnBT1

For facilitating the interconversion of CO₂ and HCO₃⁻ which is beneficial to the precipitation of CaCO₃, a plasmid containing Carbonic anhydrase coding gene have built and transferred into B. subtilis strain WB800 and express successfully.The promoter P43 used in the plasmid is a strong promoter in Bacillus subtilis 168,and the Carbonic anhydrase is from The polyextremophilic bacterium Bacillus halodurans TSLV1 (MTCC 10961, 16S rDNA Acc. No. HQ235051) which belong to α-type in classes of CAs. The terminator we used is rrnBT1(BBa_B0010) from E. coli rrnB, which will stop the Transcription of gene and raise the efficiency of expression.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 851
  Illegal NotI site found at 1138
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 831
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 376
  Illegal SapI.rc site found at 534
  Illegal SapI.rc site found at 846
  

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