Difference between revisions of "Part:BBa K2232000"
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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. | 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. | ||
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Revision as of 00:42, 6 October 2018
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. 1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 540
Illegal NotI site found at 827 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 520
- 1000INCOMPATIBLE 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).
The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining(Fig.2).
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.