View source for Part:BBa K2232000


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<partinfo>BBa_K2232000 short</partinfo>

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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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2232000 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K2232000 parameters</partinfo>
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==User Reviews==
===Contribution from iGEM2022 ZJU-China===
Group: ZJU-China 2022
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Author: Junyi Liu, Jiachen Li
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Summary: In 2022, ZJU-China used this part in their project, experimentally characterized the function of the carbonic anhydrase encoded by this part.We also performed codon optimization and compared the function of the parts before and after optimization.

===Characterization===
We've created Part:<b><partinfo>BBa_K4202004</partinfo></b> ,which is improved by codon optimization from Part:<b><partinfo>BBa_K2232000</partinfo></b>. Then we have done a series of control experiments to compare the function of two parts. Both of the two parts encodes carbonic anhydrase, so we name the carbonic anhydrase expressed by  <b><partinfo>BBa_K2232000</partinfo></b> as <b>CA1</b>, while name the carbonic anhydrase expressed by <b><partinfo>BBa_K4202004</partinfo></b> as <b>CA2</b>.
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===Expression of BBa_K2232000===
After chemical transformation of plasmid with this part to <i>Basillus subtilis</i> WB600, the transformed <i>Bacillus subtilis</i> WB600 were cultured in optimized LB and SMM medium, and obtained crude enzyme solution by centrifugation and ultrasonic disruption.Then we detected the molecular mass by SDS-PAGE and coomassie blue staining.
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SDS-PAGE displayed bands of 37kDa and 74kDa for CA monomer and dimer, which didn' t exist in the control group(Fig.1-1).
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<div align="center">[[File:Liu Junyi 1-1.png]]</div>
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<div align="center"><b>Fig 1-1</b> SDS-PAGE and Coomassie brilliant blue staining results of whole protein lysates of strain CA1, strain CA2 and blank WB600. Lane 1: Protein Ladder; Lane2: CA2 strain grown in LB medium, Lane3: CA2 strain grown in SMM medium, Lane4: CA1 strain grown in LB medium, Lane5: CA1 strain grown in SMM medium, Lane6: blank WB600 strain.</div>
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===Determination the ability of TSLV-BS-CA to hydrate CO<sub>2</sub>===
<p>To measure the activity of CA, we used the modified Wilbur-Anderson's method. As we know, CA can catalyze CO<sub>2</sub> hydration and at the same time release H<sup>+</sup> reducing the pH. According to that, we chose bromothymol blue, an acid-base indicator that appears yellow when pH≤6 and blue when pH > 7.6. Therefore, the color development of bromothymol blue can indirectly reflect the change of pH from 8.0 to 6.0 by CA. </p>
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<p>After adding ice-saturated CO<sub>2</sub> solution for 10min, the color of the tubes containing crude enzyme solution CA1 and CA2 began to change, indicating that the pH of the solution began to decrease. After 10min, the tubes containing the crude enzyme solution showed significant discoloration, and after 5 days, the tubes containing the CA1 crude enzyme solution turned completely yellow, implying that the pH had decreased from 8.0 to 6.0 due to the formation of H<sup>+</sup> during CO<sub>2</sub> hydration(Fig.1-3).</p>
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<p>The activity of CA1 and CA2 were verified in this experiment, but the enzyme activities were weak, possibly due to low enzyme expression or insufficient concentration of unpurified enzyme. In addition, the catalytic rate of CA2 crude enzyme solution was lower than that of CA1.</p>
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<div align="center">[[File:Liu Junyi 1-3.png|800px|]]</div>
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<div align="center"><b>Fig 1-3</b> The activity of CA detected by the Wilbur-Anderson's method. From left to right, the four tubes were pH=6.0 Tris-HCl buffer with bromothymol blue indicator, reaction system with blank WB600 lysate, CA2 crude enzyme solution, CA1 crude enzyme solution. A: Initial reaction solution（0min); B: 10min after adding ice-saturated CO<sub>2</sub> solution; C: 5d after adding ice-saturated CO<sub>2</sub> solution.</div>
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===Determination the ability of TSLV-BS-CA to precipitate CaCO<sub>3</sub>===
<p>To test the ability of engineered <i>Bacillus subtilis</i> WB600 to precipitate CaCO<sub>3</sub>, we cultured the engineered bacteria in 30ml of LB medium at 25℃ for 3 days and added 5 ml of 100mM CaCl<sub>2</sub> solution on the first and second days. We filtered the culture medium through a Whatman membrane filter paper to separate the bacteria and CaCO<sub>3</sub>. The bacteria and CaCO<sub>3</sub> were dried and weighed, respectively. We can calculate  CaCO<sub>3</sub> precipitation capacity of engineered bacteria by the formula:CaCO<sub>3</sub> dry weight (mg)/cell dry weight (g).</p>
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<p>After three days of cultivation, we could clearly see that the culture medium of the CA1 and CA2 transformant became turbid(Fig.1-4 A). The precipitate was filtered and dried.(Fig.1-4B). According to the the foemula:CaCO<sub>3</sub> production capacity = CaCO<sub>3</sub> dry weight (mg)/cell dry weight (g), we  found that the the precipitation efficiency of CA1 was higher than that of CA2(Fig.1-4C).
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<div align="center">[[File:Liu Junyi 1-5.png|800px|]]</div>
<div align="center"><b>Fig 1-4</b> A: The turbidity of the culture solution. B: Filtered and dried sediment products. C: CaCO<sub>3</sub> productions by CA1 and CA2 for 3d.</div>
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==User Reviews==
Yuru_chen<br>
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  (<partinfo>BBa_K2232000</partinfo>).<br>
===Contribution from iGEM2018 AHUT_China===
<p>The sequence of  (<partinfo>BBa_K2232000</partinfo>) was synthesized and cloned into the expression plasmid pET-30a(+) to obtain the recombinant expression vector (Fig. 1).</p>     
<div align="center">https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_comment22.jpg</div>

<center>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.
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<p>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  (<partinfo>BBa_K2232000</partinfo>) can be expressed in our chassis E. coli BL21 (DE3).</p>
<div align="center">https://static.igem.org/mediawiki/parts/0/0f/T--AHUT_China--_comment11.jpg</div>
<center>Fig. 2 Western blot analysis of protein extracted from lysates of TSLV1-CA expressed E.coli BL21(DE3) strain
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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). 
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<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>
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The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining(Fig.2).
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<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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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.
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<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
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