Difference between revisions of "Part:BBa K5530002"

Latest revision as of 07:18, 29 September 2024

pET28a-PbCBM56-mcherry

Sequence and Features

Construction Design

CBM56 is the affinity protein for β-1,3-glucan, which is a main component of the fungal cell wall. This protein binds to fungal cell walls with a certain concentration of β-1,3-glucan. mCherry is a protein with red fluorescence. The fusion probe is used to mark the fungi after CBM56-mCherry attaches to the cell wall (Figure 1). The CBM56 gene sequence was obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/), using the pET28a plasmid.

Figure 1: The plasmid map of pET-CBM56-mCherry

Engineering Principle

80% of the dry weight of the fungal cell wall consists of carbohydrates, including β-1,3-glucan, chitin, deacetylated chitosan, cellulose, and galactomannan. In this study, a fungal-specific visual detection probe was constructed by fusing CBM proteins with specific affinities for β-1,3-glucan and chitin (Figure 2), enabling 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

Both gene sequences were amplified by PCR, confirmed by bands of approximately 300 bp and 700 bp on the electrophoretic gel (Figure 3. A). The gene length of CBM56 was 333 bp, and the gene length of mCherry was 737 bp, indicating successful amplification of the target bands. The linearized vector pET28a is 5320 bp. Figure 3.B shows that the plasmid was successfully linearized.

Figure 3: The electrophoretic gel map

The pET-CBM56-mCherry plasmid was built using homologous recombination and introduced into E.coli DH5α through heat shock. Successfully transfected bacterial monoclonal colonies were selected using LB-agar plates containing Kanamycin (Figure 4.B). Plasmid extraction and sequencing confirmed the successful ligation of CBM56, as shown in Figure 4A. The comparison of sequencing results in Figure 4C showed the target fragments of CBM56 and mCherry, proving that pET-CBM56-mCherry was successfully constructed.

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

Figure 5 shows the SDS-PAGE results. Figure 5B displays the purified protein supernatant, while Figure 5A displays the protein precipitate mixture. The bands corresponding to pET-CBM56-mCherry proteins, ranging from 34 kDa to 43 kDa, confirm successful expression in both the supernatant and precipitate.

Figure 5: SDS-PAGE of the pET-CBM56-mCherry(35.6kDa), pET-CBM2-mCherry(35.9kDa), and pET-CBM56-CBM2-mCherry(40.1.6kDa)

The pellet of bacterial cultures containing recombinant plasmids all showed red in color (Figure 6A). The fluorescence intensity of pET-CBM56-mCherry in Figure 6B was higher than the control group, indicating that the target gene fused with the reporter gene was successfully expressed.

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 the fluorescence intensity of Saccharomyces cerevisiae (AQ), Pichia pastoris (GS115), and Saccharomyces cerevisiae (CCTCC M94055) increased as the concentration of pET-CBM56-mCherry probes increased. The fluorescent intensity reached a maximum when the probe concentration reached 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-mCherry probes.

2. Binding effect under microscope

Figures 8 A. B. C show images of the probe pET-CBM56-mCherry combined with three different fungi, Saccharomyces cerevisiae (AQ), Pichia pastoris (GS115), and Saccharomyces cerevisiae(CCTCC M94055), under an ordinary optical microscope (10x40) and a fluorescence microscope. Under the fluorescence microscope, we can see red fluorescent dots, indicating that the pET-CBM56-mCherry probes successfully bind to the fungi. This demonstrates that the method of using fluorescent protein mCherry to detect fungi is effective.

Figure 8. Binding of pET-CBM56-mCherry to Saccharomyces cerevisiae AQ, Pichia pastoris, and Saccharomyces cerevisiae(CCTCC M94055) Figure A: Left - Optical microscope (10x40) view; Right - pET-CBM56-mCherry binding with Saccharomyces cerevisiae AQ; Figure B: Left - Optical microscope (10x40) view; Right - pET-CBM56-mCherry binding with Saccharomyces cerevisiae(CCTCC M94055); Figure C: Left - Optical microscope (10x40) view; Right - pET-CBM56-mCherry binding with Pichia pastoris GS115

References

[1] Hussain K, Malavia D, Johnson EM, 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