Difference between revisions of "Part:BBa K5530004"

 
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<partinfo>BBa_K5530004 short</partinfo>
 
<partinfo>BBa_K5530004 short</partinfo>
  
 
pET28a-PbCBM56--ChBD3-mcherry
 
pET28a-PbCBM56--ChBD3-mcherry
  
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===Usage and Biology===
 
  
 
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===Functional Parameters===
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<head>
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<partinfo>BBa_K5530004 parameters</partinfo>
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    <meta charset="UTF-8">
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<!-- -->
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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    <title>CBM56-CBM2-mCherry Probe Construction</title>
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</head>
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<body>
 +
 
 +
    <!-- Construction Design Section -->
 +
    <h2>Construction Design</h2>
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    <p>We found the gene sequence CBM56 (<a href="https://static.igem.wiki/teams/5530/bba-k5530000" target="_blank">BBa_K5530000</a>) and CBM2 (<a href="https://static.igem.wiki/teams/5530/bba-k5530001" target="_blank">BBa_K5530001</a>) on the NCBI website and used the pET28a plasmid (<a href="https://static.igem.wiki/teams/5530/bba-k3521004" target="_blank">BBa_K3521004</a>). Next, we located the mCherry fluorescent protein and used homologous recombination to create the pET-CBM56-CBM2-mCherry construct (Figure 1).</p>
 +
 
 +
    <!-- Figure 1 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/1.png" width="50%" alt="Figure 1: Map of the Recombinant Plasmid pET-CBM56-CBM2-mCherry">
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        <div style="text-align:center;">
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            <caption>Figure 1: Map of the Recombinant Plasmid pET-CBM56-CBM2-mCherry</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- Engineering Principle Section -->
 +
    <h3>Engineering Principle</h3>
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    <p>80% of the dry weight of the fungal cell wall is composed of carbohydrates, mainly including: β-1,3-glucan, chitin, deacetylated chitosan, cellulose, galactomannan, etc. In this study, a fungal-specific visual detection probe was constructed using CBM proteins with specific affinities for β-1,3-glucan and chitin (Figure 2). This allows for efficient and rapid visual detection of fungi in food safety and medical testing [1].</p>
 +
 
 +
    <!-- Figure 2 -->
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    <div style="text-align:center;">
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        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/2.png" width="50%" alt="Figure 2: Composition diagram of fungal cell wall">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 2: Composition diagram of fungal cell wall [2]</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- Cultivation, Purification, and SDS-PAGE Section -->
 +
    <h3>Cultivation, Purification, and SDS-PAGE</h3>
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    <p>To combine the two probes mentioned above, the binding proteins and the reporter protein were designed to fuse together, following the same strategy of plasmid construction. All three genes were inserted into the new plasmid, validated by DNA gel electrophoresis (Figure 3A, 3B).</p>
 +
 
 +
    <!-- Figure 3 -->
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    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/3.png" width="50%" alt="Figure 3: The electrophoretic gel map">
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        <div style="text-align:center;">
 +
            <caption>Figure 3: The electrophoretic gel map</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <p>Figure 4A shows that CBM-mCherry was successfully amplified. Colonies and sequencing results both proved that the plasmid was built as designed (Figure 4B, 4C).</p>
 +
 
 +
    <!-- Figure 4 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/4.png" width="50%" alt="Figure 4: Monoclonal colony validation and sequencing">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 4: A: Electrophoretic map of monoclonal colony validation. B: Monoclonal colony diagram of bacteria containing pET-CBM56-CBM2-mCherry. C: Plasmid sequencing map of pET-CBM56-CBM2-mCherry</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <p>Figure 5 shows the SDS-PAGE results. Figure 5A shows the SDS-PAGE of the purified protein supernatant, while Figure 5B displays the SDS-PAGE of the protein precipitate mixture. In both figures, the bands corresponding to pET-CBM56-CBM2-mCherry proteins, ranging from 34 kDa to 43 kDa, are notably intense, confirming successful expression in both the supernatant and precipitate.</p>
 +
 
 +
    <!-- Figure 5 -->
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    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/5.png" width="50%" alt="Figure 5: SDS-PAGE results of pET-CBM56-mCherry, pET-CBM2-mCherry, and pET-CBM56-CBM2-mCherry">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 5: SDS-PAGE results of pET-CBM56-mCherry, pET-CBM2-mCherry, and pET-CBM56-CBM2-mCherry</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <p>There is protein deposition in Figure 6A in the tube of pET-CBM56-CBM2-mCherry, which means it expressed successfully.</p>
 +
 
 +
    <!-- Figure 6 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/6.png" width="50%" alt="Figure 6: Fluorescent intensity of bacterial cultures">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 6: A: Bacterial Pellet of <i>E.coli</i> BL21(DE3) transfected with recombinant plasmids. B: Fluorescent Intensity of Bacterial Culture.</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- Characterization/Measurement Section -->
 +
    <h3>Characterization/Measurement</h3>
 +
 
 +
    <!-- Fluorescence Intensity Measurement -->
 +
    <h4>1. Fluorescence intensity to evaluate the detection effect of the probes</h4>
 +
    <p>Figure 7 shows that as the concentration of the pET-CBM56-CBM2-mCherry probe increases, the fluorescence intensity of <i>Saccharomyces cerevisiae</i> (AQ), <i>Pichia pastoris</i> (GS115), and <i>Saccharomyces cerevisiae</i> (CCTCC M94055) also increases. This suggests that these probes emit red fluorescence upon binding with the fungal cell wall, and the binding strength improves with higher probe concentrations. Optimal binding between the fungi and the pET-CBM56-CBM2-mCherry probe is achieved at a concentration of 3.0 mg/ml.</p>
 +
 
 +
    <!-- Figure 7 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/7.png" width="50%" alt="Figure 7: Fluorescence intensity of fungi after binding with probes">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 7: Fluorescence intensity of <i>Saccharomyces cerevisiae</i> (AQ), <i>Pichia pastoris</i> (GS115), and <i>Saccharomyces cerevisiae</i> (CCTCC M94055) after binding with different concentrations of pET-CBM56-CBM2-mCherry probes</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- Binding Effect Under Microscope -->
 +
    <h4>2. Binding effect under microscope</h4>
 +
    <p>Figures 8A, 8B, and 8C display images of the pET-CBM56-CBM2-mCherry probe combined with three fungi, <i>Saccharomyces cerevisiae</i> (AQ), <i>Pichia pastoris</i> (GS115), and <i>Saccharomyces cerevisiae</i> (CCTCC M94055), captured under both an optical microscope (10x40) and a fluorescence microscope. Red fluorescent dots are visible under the fluorescence microscope, indicating successful binding of the pET-CBM56-CBM2-mCherry probes to the fungi. This underscores the effectiveness of using the fluorescent protein mCherry for fungi detection.</p>
 +
 
 +
    <!-- Figure 8 -->
 +
    <div style="text-align:center;">
 +
        <img src="https://static.igem.wiki/teams/5530/bba-k5530004/8.png" width="50%" alt="Figure 8: Microscopy images of CBM56-CBM2-mCherry probe binding to fungi">
 +
        <div style="text-align:center;">
 +
            <caption>Figure 8: A, B, C: Binding of pET-CBM56-CBM2-mCherry to <i>Saccharomy html
 +
ces cerevisiae</i> AQ, <i>Pichia pastoris</i> GS115, and <i>Saccharomyces cerevisiae</i> (CCTCC M94055). Left: Optical microscope (10x40); Right: Fluorescence microscope.</caption>
 +
        </div>
 +
    </div>
 +
 
 +
    <!-- References Section -->
 +
    <h3>References</h3>
 +
    <ol>
 +
        <li>K. Hussain K, Malavia D, M. Johnson E, Littlechild J, Winlove CP, Vollmer F, et al. Biosensors and Diagnostics for Fungal Detection. <i>J Fungi (Basel)</i>. 2020;6: 349. doi:<a href="https://doi.org/10.3390/jof6040349" target="_blank">10.3390/jof6040349</a></li>
 +
        <li>Structure of CBM56. Available: <a href="http://www.cazy.org/CBM56_structure.html" target="_blank">http://www.cazy.org/CBM56_structure.html</a></li>
 +
    </ol>
 +
 
 +
 
 +
</body>
 +
</html>

Revision as of 11:58, 28 September 2024


pET28a-PbCBM56--ChBD3-mcherry(pET28a-CBM56--CBM2-mcherry)

pET28a-PbCBM56--ChBD3-mcherry


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 4713
    Illegal NotI site found at 5054
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4402
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2622
    Illegal NgoMIV site found at 2782
    Illegal NgoMIV site found at 4370
  • 1000
    COMPATIBLE WITH RFC[1000]


CBM56-CBM2-mCherry Probe Construction

Construction Design

We found the gene sequence CBM56 (BBa_K5530000) and CBM2 (BBa_K5530001) on the NCBI website and used the pET28a plasmid (BBa_K3521004). Next, we located the mCherry fluorescent protein and used homologous recombination to create the pET-CBM56-CBM2-mCherry construct (Figure 1).

Figure 1: Map of the Recombinant Plasmid pET-CBM56-CBM2-mCherry
Figure 1: Map of the Recombinant Plasmid pET-CBM56-CBM2-mCherry

Engineering Principle

80% of the dry weight of the fungal cell wall is composed of carbohydrates, mainly including: β-1,3-glucan, chitin, deacetylated chitosan, cellulose, galactomannan, etc. In this study, a fungal-specific visual detection probe was constructed using CBM proteins with specific affinities for β-1,3-glucan and chitin (Figure 2). This allows for efficient and rapid visual detection of fungi in food safety and medical testing [1].

Figure 2: Composition diagram of fungal cell wall
Figure 2: Composition diagram of fungal cell wall [2]

Cultivation, Purification, and SDS-PAGE

To combine the two probes mentioned above, the binding proteins and the reporter protein were designed to fuse together, following the same strategy of plasmid construction. All three genes were inserted into the new plasmid, validated by DNA gel electrophoresis (Figure 3A, 3B).

Figure 3: The electrophoretic gel map
Figure 3: The electrophoretic gel map

Figure 4A shows that CBM-mCherry was successfully amplified. Colonies and sequencing results both proved that the plasmid was built as designed (Figure 4B, 4C).

Figure 4: Monoclonal colony validation and sequencing
Figure 4: A: Electrophoretic map of monoclonal colony validation. B: Monoclonal colony diagram of bacteria containing pET-CBM56-CBM2-mCherry. C: Plasmid sequencing map of pET-CBM56-CBM2-mCherry

Figure 5 shows the SDS-PAGE results. Figure 5A shows the SDS-PAGE of the purified protein supernatant, while Figure 5B displays the SDS-PAGE of the protein precipitate mixture. In both figures, the bands corresponding to pET-CBM56-CBM2-mCherry proteins, ranging from 34 kDa to 43 kDa, are notably intense, confirming successful expression in both the supernatant and precipitate.

Figure 5: SDS-PAGE results of pET-CBM56-mCherry, pET-CBM2-mCherry, and pET-CBM56-CBM2-mCherry
Figure 5: SDS-PAGE results of pET-CBM56-mCherry, pET-CBM2-mCherry, and pET-CBM56-CBM2-mCherry

There is protein deposition in Figure 6A in the tube of pET-CBM56-CBM2-mCherry, which means it expressed successfully.

Figure 6: Fluorescent intensity of bacterial cultures
Figure 6: A: Bacterial Pellet of E.coli BL21(DE3) transfected with recombinant plasmids. B: Fluorescent Intensity of Bacterial Culture.

Characterization/Measurement

1. Fluorescence intensity to evaluate the detection effect of the probes

Figure 7 shows that as the concentration of the pET-CBM56-CBM2-mCherry probe increases, the fluorescence intensity of Saccharomyces cerevisiae (AQ), Pichia pastoris (GS115), and Saccharomyces cerevisiae (CCTCC M94055) also increases. This suggests that these probes emit red fluorescence upon binding with the fungal cell wall, and the binding strength improves with higher probe concentrations. Optimal binding between the fungi and the pET-CBM56-CBM2-mCherry probe is achieved at a concentration of 3.0 mg/ml.

Figure 7: Fluorescence intensity of fungi after binding with probes
Figure 7: Fluorescence intensity of Saccharomyces cerevisiae (AQ), Pichia pastoris (GS115), and Saccharomyces cerevisiae (CCTCC M94055) after binding with different concentrations of pET-CBM56-CBM2-mCherry probes

2. Binding effect under microscope

Figures 8A, 8B, and 8C display images of the pET-CBM56-CBM2-mCherry probe combined with three fungi, Saccharomyces cerevisiae (AQ), Pichia pastoris (GS115), and Saccharomyces cerevisiae (CCTCC M94055), captured under both an optical microscope (10x40) and a fluorescence microscope. Red fluorescent dots are visible under the fluorescence microscope, indicating successful binding of the pET-CBM56-CBM2-mCherry probes to the fungi. This underscores the effectiveness of using the fluorescent protein mCherry for fungi detection.

Figure 8: Microscopy images of CBM56-CBM2-mCherry probe binding to fungi
Figure 8: A, B, C: Binding of pET-CBM56-CBM2-mCherry to Saccharomy html ces cerevisiae AQ, Pichia pastoris GS115, and Saccharomyces cerevisiae (CCTCC M94055). Left: Optical microscope (10x40); Right: Fluorescence microscope.

References

  1. K. Hussain K, Malavia D, M. Johnson E, Littlechild J, Winlove CP, Vollmer F, et al. Biosensors and Diagnostics for Fungal Detection. J Fungi (Basel). 2020;6: 349. doi:10.3390/jof6040349
  2. Structure of CBM56. Available: http://www.cazy.org/CBM56_structure.html