Difference between revisions of "Part:BBa K4863005"
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hpCA is a metalloenzymes responsible for catalyzing the interconversion of carbon dioxide (CO2) to bicarbonate ions (HCO3−). It is an α-carbonic anhydrase isolated from the bacterium <i>Helicobacter pylori</i> 26695, codon optimized for expression in <i>E. Coli</i> BL21(DE3). Slp forms the paracrystalline S-layer by forming a regular lattice through an entropy-driven process. It has an undefined signal peptide at the C-terminus.  
 
hpCA is a metalloenzymes responsible for catalyzing the interconversion of carbon dioxide (CO2) to bicarbonate ions (HCO3−). It is an α-carbonic anhydrase isolated from the bacterium <i>Helicobacter pylori</i> 26695, codon optimized for expression in <i>E. Coli</i> BL21(DE3). Slp forms the paracrystalline S-layer by forming a regular lattice through an entropy-driven process. It has an undefined signal peptide at the C-terminus.  
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===Characterization===
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To achieve the surface display of hpCA on <i>Synechocystis</i> PCC 6803, hpCA is fused with SpyCatcher via a flexible linker so that the covalent bonding between SpyCatcher and SpyTag displayed on the surface of <i>Synechocystis</i> PCC 6803 would anchor hpCA to the cell.
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We designed the plasmid pKeystone013 to express hpCA-SpyCatcher in <i>E. coli</i> BL21 (DE3). hpCA is fused with a modified SpyCatcher via a 21-bp flexible linker. This plasmid is driven by a T7 promoter and controlled by a lac repressor and a lac operon, which regulate the gene expression with response to IPTG supply.
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We transformed the plasmid into <i>E. coli</i> DH5𝛼 for plasmid amplification in large quantities. The gel electrophoresis of the colony PCR product of the transformed strain indicates successful construction of the plasmid, which is also confirmed by the sequencing result. 
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<p style="font-size: smaller; margin-top: 10px;"> Fig.1 Gel electrophoresis verification of the hpCA-SpyCatcher sequence after plasmid transformation into <i>E. coli</i> DH5𝛼. The primers used for colony PCR correspond to a 1359 bp-long product, which is close to the 1200-bp bands observed. </p>
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We then transformed the plasmid into <i>E. coli</i> BL21 (DE3) for hpCA-SpyCatcher expression. Only the bacteria treated with IPTG during culture can activate the expression of hpCA-SpyCatcher. SDS-PAGE result of the protein purified from engineered <i>E. coli</i> BL21 (DE3) demonstrate successful expression.
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<p style="font-size: smaller; margin-top: 10px;"> Fig.2: SDS-PAGE verification of the extracted hpCA-SpyCatcher protein complex, showing a band near 40-kDA (hpCA-SpyCatcher has a molecular weight of 40.5 kDa). </p>
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An enzyme activity assay was conducted for the purified protein complex using the colorimetric method. The significant difference between the catalytic activity of hpCA-SpyCatcher (≥0.04) and the control (≤0.02) demonstrates the substantive enzyme activity of our purified hpCA-SpyCatcher.
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<p style="font-size: smaller; margin-top: 10px;"> Fig.3: hpCA-SpyCatcher enzyme activity assay. The control used was Tris-HCl. </p>
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Since hpCA efficiently catalyzes the hydration reaction H2O + CO2⇌HCO3− + H in which HCO3− further reacts with CaCl2 to form CaCO3, some white precipitation and suspension can be observed in the flask with hpCA-SpyCatcher solution after a period of time. We infer that the white solid formed is calcium carbonate which arises from the reaction induced by hpCA. In contrast, the control solution does not undergo observable chemical change during the process. This experiment indicates that the catalytic activity of hpCA-SpyCatcher can be reflected by real experiment result and calcium carbonate precipitation is likely to be achieved by using hpCA. 
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<p style="font-size: smaller; margin-top: 10px;"> Fig.4: Induction of calcium carbonate precipitation by hpCA-SpyCatcher complex (labeled “CAS”). Control is pure water. </p>
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Latest revision as of 12:39, 11 October 2023


T7_hpCA_Slp

alpha type carbonic anhydrase fused with s-layer protein via a flexible linker for surface display of hpCA on the cell surface of Synechocystis PCC6803. This yields a cell completely covered in the fusion protein. Surface display maximizes hpCA's binding to substrate and ensures its stability.

Usage and Biology

The fusion of a protein to the Slp N-terminus has proved to be an effective system for the functional surface display of cyanobacteria. For the project of this year, we displayed α-carbonic anhydrase hpCA on the Synechocystis PCC6803 cell surface with the fusion with Slp on the cell surface for the production of calcium carbonate precipitates.

hpCA is a metalloenzymes responsible for catalyzing the interconversion of carbon dioxide (CO2) to bicarbonate ions (HCO3−). It is an α-carbonic anhydrase isolated from the bacterium Helicobacter pylori 26695, codon optimized for expression in E. Coli BL21(DE3). Slp forms the paracrystalline S-layer by forming a regular lattice through an entropy-driven process. It has an undefined signal peptide at the C-terminus.

Characterization

To achieve the surface display of hpCA on Synechocystis PCC 6803, hpCA is fused with SpyCatcher via a flexible linker so that the covalent bonding between SpyCatcher and SpyTag displayed on the surface of Synechocystis PCC 6803 would anchor hpCA to the cell.

We designed the plasmid pKeystone013 to express hpCA-SpyCatcher in E. coli BL21 (DE3). hpCA is fused with a modified SpyCatcher via a 21-bp flexible linker. This plasmid is driven by a T7 promoter and controlled by a lac repressor and a lac operon, which regulate the gene expression with response to IPTG supply.

We transformed the plasmid into E. coli DH5𝛼 for plasmid amplification in large quantities. The gel electrophoresis of the colony PCR product of the transformed strain indicates successful construction of the plasmid, which is also confirmed by the sequencing result.

Fig.1 Gel electrophoresis verification of the hpCA-SpyCatcher sequence after plasmid transformation into E. coli DH5𝛼. The primers used for colony PCR correspond to a 1359 bp-long product, which is close to the 1200-bp bands observed.

We then transformed the plasmid into E. coli BL21 (DE3) for hpCA-SpyCatcher expression. Only the bacteria treated with IPTG during culture can activate the expression of hpCA-SpyCatcher. SDS-PAGE result of the protein purified from engineered E. coli BL21 (DE3) demonstrate successful expression.

Fig.2: SDS-PAGE verification of the extracted hpCA-SpyCatcher protein complex, showing a band near 40-kDA (hpCA-SpyCatcher has a molecular weight of 40.5 kDa).

An enzyme activity assay was conducted for the purified protein complex using the colorimetric method. The significant difference between the catalytic activity of hpCA-SpyCatcher (≥0.04) and the control (≤0.02) demonstrates the substantive enzyme activity of our purified hpCA-SpyCatcher.

Fig.3: hpCA-SpyCatcher enzyme activity assay. The control used was Tris-HCl.

Since hpCA efficiently catalyzes the hydration reaction H2O + CO2⇌HCO3− + H in which HCO3− further reacts with CaCl2 to form CaCO3, some white precipitation and suspension can be observed in the flask with hpCA-SpyCatcher solution after a period of time. We infer that the white solid formed is calcium carbonate which arises from the reaction induced by hpCA. In contrast, the control solution does not undergo observable chemical change during the process. This experiment indicates that the catalytic activity of hpCA-SpyCatcher can be reflected by real experiment result and calcium carbonate precipitation is likely to be achieved by using hpCA.

Fig.4: Induction of calcium carbonate precipitation by hpCA-SpyCatcher complex (labeled “CAS”). Control is pure water.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 443
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference