Difference between revisions of "Part:BBa K2232014"
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<partinfo>BBa_K2232014 parameters</partinfo> | <partinfo>BBa_K2232014 parameters</partinfo> | ||
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| + | ===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 CO<sub>2</sub> to produce HCO<sub>3</sub><sup>-</sup> and captures free Ca<sup>2+</sup> with OH<sup>-</sup> 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). | ||
| + | <div> | ||
| + | <center><html><img src='https://static.igem.org/mediawiki/parts/d/d0/TSLV1-CA1.jpeg' style="width:30%;margin:0 auto"> | ||
| + | <center>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.</center> | ||
| + | </html></center> | ||
| + | </div> | ||
| + | |||
| + | The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining(Fig.2). | ||
| + | <div> | ||
| + | <center><html><img src='https://static.igem.org/mediawiki/parts/5/50/TSLV1-CA2.png' style="width:30%;margin:0 auto"> | ||
| + | <center>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). | ||
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| + | </center> | ||
| + | </html></center> | ||
| + | </div> | ||
| + | |||
| + | For determining the activity of CA, hydration of CO<sub>2</sub> 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 CO<sub>2</sub> 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. | ||
| + | <div> | ||
| + | <center><html><img src='https://static.igem.org/mediawiki/parts/2/2c/TSLV1-CA4.png' style="width:50%;margin:0 auto"> | ||
| + | <center>Fig.3 CA activity of crude enzyme solution from measured by Brownell’s method | ||
| + | </center> | ||
| + | </html></center> | ||
| + | </div> | ||
Revision as of 02:57, 2 November 2017
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 851
Illegal NotI site found at 1138 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 831
- 1000INCOMPATIBLE 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).
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.
