Part:BBa_K5530004

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]

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

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 [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).Fig.3 showed that the target gene was consistent with the size of the band, indicating that the PCR amplification was successful.

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: 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 and pET-CBM56-CBM2-mCherry(40.1kDa)

There is red protein deposition in Figure 6A in the tube of pET-CBM56-CBM2-mCherry, which means it expressed successfully.The fluorescence intensity of pET-CBM56-CBM2 mcherry was significantly higher than that of the control group, which proved that the protein expression was successful.

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 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: 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