Experimental Overview
After designing the fragments and primers, we utilized Gibson assembly for DNA construction: linearized vectors and target fragments were assembled to generate circular recombinant plasmids. These assembled products were then transformed into E. coli DH5α.
Subsequently, we performed a series of functional verification experiments divided into multiple modules:
- In the signal peptide function test, methods like fluorescence observation and fluorescence detection were used
- For the chitinase activity test, techniques including SDS-PAGE and enzyme activity assays were conducted
- Within the β-amyrin synthesis module, analyses such as LC-MS were employed to determine product yield
- For the antagonistic test, approaches like fluorescence detection with microplate reader and flow cytometry were adopted to assess antagonistic effects.
Protocols
- Agarose Gel Electrophoresis
- Chemical Transformation of E. coli
- Chitinase Activity Assay
- Colony PCR
- Cryopreservation
- Gibson Assembly
- Semi-quantitative Antagonistic Assay (Extracellular Fluorescent Protein, Microplate Reader)
- Colloidal Chitin Plate Hydrolysis Assay
- Induction of Protein Expression
- LB Culture Media
- PCR
- Plasmid Extraction
- Preparation of Competent E. coli Cells
- SDS-PAGE
- Quantitative Mass Spectrometry for Amyrin
- Quantitative Antagonistic Assay (PI Stain, Flow Cytometry)
- Quantitative Secretion Efficacy Assay for Signal Peptides