Difference between revisions of "Part:BBa K5520011"

 
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<partinfo>BBa_K5520011 short</partinfo>
 
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pT7-LacO-His- CsnBP115A
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The mutant gene CsnP115A is inserted after T7 promoter, lac operator and RBS on the vector PET-28a.
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
===Functional Parameters===
 
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===Usage and Biology===
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==Plasmid construction==
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The recombinant plasmid PET-28a-CsnB was generated and utilized as the template DNA for reverse PCR using the primers  P-F-A and  P-R-A, targeting a 6073bp fragment. Subsequently, DpnI enzyme treatment was employed to remove the template DNA, following which the digested products were transformed. To confirm successful transformation, colony PCR was conducted on the transformed colonies into E. coli BL21 (DE3) using the primers CSNB-CX-F and CSNB-CX-R to amplify a 928-bp fragment. Positive colonies displaying the expected PCR product were selected for transfection. After plasmid extraction from these colonies, sequencing was performed to validate the correct insertion, resulting in the confirmation of the recombinant plasmid PET-28a-CsnBP115A.
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<h2>Test of CsnB-P115A protein</h2>
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<h3>1. SDS-PAGE</h3>
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<p>Recombinant mutant strain with PET-28a-CsnB-P115A was successfully expressed in E. coli BL21(DE3) following IPTG induction. Purification of CsnB-P115A enzyme was accomplished by Ni-NTA affinity chromatography, and both the unpurified and purified proteins were verified via SDS-PAGE. As illustrated in Figure below, distinct lane 3 was observed in the unpurified enzyme sample within the molecular weight range of 30 kDa, which corresponds to the expected theoretical value. After purification, the mutant lane 3 closely resembled that of CsnB, with a single lane detected at a position consistent with the unpurified enzyme solution.</p>
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<h3>2. Enzymatic activity determination of CsnB mutant</h3>
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<p>The DNS method was used to detect the enzyme activity of CsnB. The standard curve was plotted using the concentration of glucosamine and OD540 as the horizontal and vertical coordinates, respectively (Figure a). The enzyme activity of CsnB and its mutants was determined as shown in the figure below. The wild-type enzyme exhibits an activity of 28.8 (U/mL), while the P115A mutant shows a 15.2% increase in chitosanase activity.</p>
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<h3>3. Product analysis of mutant enzymes</h3>
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<p>CsnB and its mutants were incubated with 0.5% colloidal chitosan in an acetic acid-sodium acetate buffer at 50°C and pH 6 for 24 hr. </p>
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<h3>4. Analysis of Enzyme Activity and Product Change Mechanisms</h3>
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<p>As for the P115A mutant, the original proline residue, due to its strong rigidity, may interfere with the proper binding of the substrate. Mutating P115 to alanine increases the flexibility of the unstructured coil region, bringing the enzyme closer to the substrate. This mutation improves the enzyme's ability to bind to the substrate, thereby enhancing its catalytic activity.</p>
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Revision as of 06:25, 30 September 2024


pT7-LacO-His- CsnBP115A

The mutant gene CsnP115A is inserted after T7 promoter, lac operator and RBS on the vector PET-28a.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 844
    Illegal NotI site found at 804
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 813
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 153
    Illegal AgeI site found at 636
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Plasmid construction

The recombinant plasmid PET-28a-CsnB was generated and utilized as the template DNA for reverse PCR using the primers P-F-A and P-R-A, targeting a 6073bp fragment. Subsequently, DpnI enzyme treatment was employed to remove the template DNA, following which the digested products were transformed. To confirm successful transformation, colony PCR was conducted on the transformed colonies into E. coli BL21 (DE3) using the primers CSNB-CX-F and CSNB-CX-R to amplify a 928-bp fragment. Positive colonies displaying the expected PCR product were selected for transfection. After plasmid extraction from these colonies, sequencing was performed to validate the correct insertion, resulting in the confirmation of the recombinant plasmid PET-28a-CsnBP115A.

Test of CsnB-P115A protein

1. SDS-PAGE

Recombinant mutant strain with PET-28a-CsnB-P115A was successfully expressed in E. coli BL21(DE3) following IPTG induction. Purification of CsnB-P115A enzyme was accomplished by Ni-NTA affinity chromatography, and both the unpurified and purified proteins were verified via SDS-PAGE. As illustrated in Figure below, distinct lane 3 was observed in the unpurified enzyme sample within the molecular weight range of 30 kDa, which corresponds to the expected theoretical value. After purification, the mutant lane 3 closely resembled that of CsnB, with a single lane detected at a position consistent with the unpurified enzyme solution.

2. Enzymatic activity determination of CsnB mutant

The DNS method was used to detect the enzyme activity of CsnB. The standard curve was plotted using the concentration of glucosamine and OD540 as the horizontal and vertical coordinates, respectively (Figure a). The enzyme activity of CsnB and its mutants was determined as shown in the figure below. The wild-type enzyme exhibits an activity of 28.8 (U/mL), while the P115A mutant shows a 15.2% increase in chitosanase activity.


3. Product analysis of mutant enzymes

CsnB and its mutants were incubated with 0.5% colloidal chitosan in an acetic acid-sodium acetate buffer at 50°C and pH 6 for 24 hr.

4. Analysis of Enzyme Activity and Product Change Mechanisms

As for the P115A mutant, the original proline residue, due to its strong rigidity, may interfere with the proper binding of the substrate. Mutating P115 to alanine increases the flexibility of the unstructured coil region, bringing the enzyme closer to the substrate. This mutation improves the enzyme's ability to bind to the substrate, thereby enhancing its catalytic activity.