Difference between revisions of "Part:BBa K4202004"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4202004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4202004 SequenceAndFeatures</partinfo>
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===Usage and Biology===
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<partinfo>BBa_K4202004 parameters</partinfo>
 
<partinfo>BBa_K4202004 parameters</partinfo>
 
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===Characterization===
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As Part:<b><partinfo>BBa_K4202004</partinfo></b> is improved by codon optimization from Part:<b><partinfo>BBa_K2232000</partinfo></b>, 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_K4202004===
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After chemical transformation of plasmid with this part, 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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<br>
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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).
 +
<br>
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<div align="center">[[File:Liu Junyi 1-1.png]]</div>
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<br>
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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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===Purification of TSLV-BS-CA===
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<p>We added a 6X His tag to the N-terminus of CA on the vector, and the CA was purified by agarose-nickel column affinity chromatography. SDS-page analysis was performed on the flowing fluid, washing fluid and eluent during the purification of CA1 and CA2 by Ni-column. The eluate of CA1 and CA2 had two obvious bands at 37kDa and 74kDa, which were monomer CA and dimer CA, respectively. However, the Ni column had much non-specific  protein binding, we can add a small amount of imidazole to reduce non-specific protein binding(Fig.1-2).</p>
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<br>
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<div align="center">[[File:Liu Junyi 1-2.png]]</div>
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<p><div align="center"><b>Fig 1-2</b> A: SDS-PAGE analysis of CA1 protein purified by Ni column. Lane1: flowing fluid, Lane2-4: the 1-4 washing fluid, Lane5-9: the 1-5 eluate, Lane10: protein Maker  B:SDS-PAGE analysis of CA2 protein purified by Ni column. Lane1-5: the 1-5 eluent, Lane6-8: the 1, 2 and 4 washing fluid, Lane9: the flowing fluid, Lane10: protein Maker.</div></p>
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===Determination the ability of TSLV-BS-CA to hydrate CO<sub>2</sub>===
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<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>
 +
<br>
 +
<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>
 +
<br>
 +
<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>
 +
<br>
 +
<div align="center">[[File:Liu Junyi 1-3.png|800px|]]</div>
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<br>
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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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<br>
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===Determination the ability of TSLV-BS-CA to precipitate CaCO<sub>3</sub>===
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<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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<br>
 +
<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).
 +
<br>
 +
<div align="center">[[File:Liu Junyi 1-5.png|800px|]]</div>
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<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>

Revision as of 04:04, 26 September 2022


TSLV-BS-CA

Usage and Biology

Biomineralization can be used to deposit calcium carbonate on the surface of microbial cells, filling cracks in stone artifacts. This part is the coding sequence (CDS) of Carbonic anhydrase (CA) encoding a zinc-containing enzyme, α-carbonic anhydrase, which efficiently catalyzes the reversible hydration of CO2 to rapidly produce bicarbonate (HCO3-) and protons (H+).

Chemical reaction equation: H2O+CO2↔HCO3-+H+


Bicarbonate (HCO3-) can be transported down the concentration gradient to the outside of the cell. When we provide calcium ions (Ca2+) in the extracellular medium, the bicarbonate can combine to the Ca2+ to form calcium carbonate precipitates. In our project, the calcium carbonate precipitate can accumulate in the tiny cracks of the stone artifacts, filling the cracks and providing support.

This part derivers from BBa_K2232000 We replace codons in the original mRNA sequence with codons that are used frequently in Bacillus subtilis to ensure that the codons in the newly designed mRNA sequence are more compatible with the codon usage bias of Bacillus subtilis, avoiding the emergence of rare codons


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 540
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 166
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 535


Characterization

As Part:BBa_K4202004 is improved by codon optimization from Part:BBa_K2232000, 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 BBa_K2232000 as CA1, while name the carbonic anhydrase expressed by BBa_K4202004 as CA2.

Expression of BBa_K4202004

After chemical transformation of plasmid with this part, the transformed Bacillus subtilis 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.
SDS-PAGE displayed bands of 37kDa and 74kDa for CA monomer and dimer, which didn' t exist in the control group(Fig.1-1).

Liu Junyi 1-1.png


Fig 1-1 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.

Purification of TSLV-BS-CA

<p>We added a 6X His tag to the N-terminus of CA on the vector, and the CA was purified by agarose-nickel column affinity chromatography. SDS-page analysis was performed on the flowing fluid, washing fluid and eluent during the purification of CA1 and CA2 by Ni-column. The eluate of CA1 and CA2 had two obvious bands at 37kDa and 74kDa, which were monomer CA and dimer CA, respectively. However, the Ni column had much non-specific protein binding, we can add a small amount of imidazole to reduce non-specific protein binding(Fig.1-2).


Liu Junyi 1-2.png


Fig 1-2 A: SDS-PAGE analysis of CA1 protein purified by Ni column. Lane1: flowing fluid, Lane2-4: the 1-4 washing fluid, Lane5-9: the 1-5 eluate, Lane10: protein Maker B:SDS-PAGE analysis of CA2 protein purified by Ni column. Lane1-5: the 1-5 eluent, Lane6-8: the 1, 2 and 4 washing fluid, Lane9: the flowing fluid, Lane10: protein Maker.

Determination the ability of TSLV-BS-CA to hydrate CO2

To measure the activity of CA, we used the modified Wilbur-Anderson's method. As we know, CA can catalyze CO2 hydration and at the same time release H+ 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.


After adding ice-saturated CO2 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+ during CO2 hydration(Fig.1-3).


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.


Liu Junyi 1-3.png


Fig 1-3 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 CO2 solution; C: 5d after adding ice-saturated CO2 solution.


Determination the ability of TSLV-BS-CA to precipitate CaCO3

To test the ability of engineered Bacillus subtilis WB600 to precipitate CaCO3, we cultured the engineered bacteria in 30ml of LB medium at 25℃ for 3 days and added 5 ml of 100mM CaCl2 solution on the first and second days. We filtered the culture medium through a Whatman membrane filter paper to separate the bacteria and CaCO3. The bacteria and CaCO3 were dried and weighed, respectively. We can calculate CaCO3 precipitation capacity of engineered bacteria by the formula:CaCO3 dry weight (mg)/cell dry weight (g).


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:CaCO3 production capacity = CaCO3 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).

Liu Junyi 1-5.png
Fig 1-4 A: The turbidity of the culture solution. B: Filtered and dried sediment products. C: CaCO3 productions by CA1 and CA2 for 3d.