Notebook

Conducted literature review and finalized experimental design, including validation strategies.
Completed plasmid construction and codon optimization for compatibility with the yeast expression system.
Completed constructs:
- pGBKT7-Msn2-EGFP
- pGBKT7-FBP1-EGFP
- pESC-URA-TEF1-TAAR5

Evaluated sodium butyrate (NaB) cytotoxicity in murine microglia cell (BV2) and mouse hippocampal neuronal cell line (HT22) using the CCK-8 assay.
Performed routine cell cryopreservation and subculturing.
Seeded cells in 96-well plates and treated with NaB at graded concentrations. Cell viability was quantified via the CCK-8 assay.

Validated NaB-mediated inhibition of LPS-induced inflammatory responses in BV2 cells using the CCK-8 assay and flow cytometry.
Established the optimal NaB working concentration for subsequent experiments.

Maintained Mouse colorectal cancer epithelial cells (MCCE) through subculturing and cryopreservation.
Investigated adhesive properties of yeast expressing transadhesion protein genes to MCCE cells using confocal microscopy and flow cytometry.

Successfully generated the following plasmids:
- pGBKT7-Msn2-EGFP
- pGBKT7-FBP1-EGFP
- pESC-URA-TEF1-TAAR5

Investigated the interaction between MCEC (Mouse Colorectal Cancer Epithelial Cells) and WT as well as Syn-type Saccharomyces cerevisiae, respectively. Favorable results were obtained, with Syn-type yeast exhibiting significantly stronger adhesive capacity.
Performed expansion of plasmid-containing bacterial strains, followed by plasmid extraction and bacterial strain preservation.

Thawed and revived Mouse Hippocampal Neuronal Cell Line (HT-22) and Murine Microglial Cell Line (BV-2).
Treated BV-2 cells with sodium butyrate (NaB) and lipopolysaccharide (LPS).
Established co-cultivation systems of HT-22 cells and BV-2 cells, and conducted flow cytometry analysis.

Performed plasmid transformation of the pGBKT7 series and pESC-URA series plasmids into the Wild-Type (WT, INVSc1) Saccharomyces cerevisiae strain.
Verified the transformation results as positive via PCR, and confirmed the authenticity of the transformants through genomic DNA extraction.

Performed transformation of the Wild-Type INVSc1 Saccharomyces cerevisiae strain, and named the resulting transformants S13-2, S13-3, and S13-4 respectively.
Carried out transformation of the pGBKT7-Msn2-EGFP plasmid into yeast, and named the obtained transformants S12-7, S12-8, and S12-9 respectively.
Performed co-transformation of the pGBKT7-Fbp1-EGFP and pESC-Ura-TEF1-TAAR5 plasmids into yeast.

Conducted transformation verification for yeast transformants S12-7, S12-8, S12-9, S13-7, S13-8, and S13-9.
Using transformant S12-8 as the host strain, performed transformation of the pESC-Ura-TEF1-TAAR5 plasmid.
Using Wild-Type INVSc1 as the host strain, carried out co-transformation of the pESC-Ura-TEF1-TAAR5 and pGBKT7-Fbp1-EGFP plasmids.
Treated yeast transformants S13-8 and S13-9 with gradient concentrations of hydrogen peroxide to verify the plasmid-mediated expression and response to reactive oxygen species (ROS).

Using transformant S11-2 as the host strain, performed secondary transformation of the pESC-Ura-TEF1-TAAR5 plasmid.
Using transformant S12-8 as the host strain, performed secondary transformation of the pESC-Ura-TEF1-TAAR5 plasmid.
Using Wild-Type INVSc1 as the host strain, carried out co-transformation of the pESC-Ura-TEF1-TAAR5 and pGBKT7-Fbp1-EGFP plasmids.
Verified the co-transformation transformants via PCR.
Performed plasmid extraction and bacterial strain preservation.

Using Wild-Type INVSc1 Saccharomyces cerevisiae as the host strain, performed transformation of the pESC-Ura-TEF1-TAAR5 plasmid.

Lithium acetate-mediated co-transformation of plasmids 11+14 and 12+14 double plasmid systems
Colony PCR verification of plasmid 14 positive transformants
Lithium acetate transformation of plasmids 11, 12, and 13

Colony PCR verification of plasmid 13 positive transformants
Colony PCR verification of 12+14 positive transformants
Colony PCR verification of plasmid 14 positive transformants
Sequencing identification of transformants confirmed above results as false positives
Genomic DNA extraction from 12+14 putative positive transformants for genomic PCR analysis

Recovery and cultivation of 13-8 positive transformants on SC-Trp selective medium
Recovery and cultivation of 13-15 and 13-9 positive transformants on SC-Trp selective medium
ROS response capability assessment of 13-15, 13-9, and 13-8 positive transformants using microplate reader assays
TMA verification of signal transduction pathways and GPCR system functionality in 12+14 positive transformants
Fluorescence microscopy examination of plasmid 13 positive transformants and 12+14 positive transformants

ROS response capability assessment of 13-8 positive transformants using microplate reader assays
Data processing and analysis
Plating of E. coli harboring plasmid 16 on LB solid medium, isolation of single colonies, and cultivation in LB liquid medium for amplification
Extraction of plasmid 16 using phenol extraction method
Transformation with plasmid 16

Colony PCR verification of plasmid 14 positive transformants
Construction of hydrogen peroxide dose-response curves for reporter gene activity
Lithium acetate transformation of plasmid 16 with different plasmid concentration gradients

Flow cytometry analysis of ROS response capability in 13-8 positive transformants
Organization and compilation of experimental data
Lithium acetate transformation of plasmid 16 with extended heat shock duration
Lithium acetate transformation of plasmid 16 with increased plasmid concentration
Colony PCR verification of plasmid 16 positive transformants

Colony PCR verification of plasmid 16 positive transformants
Sequencing verification of plasmid 16 positive transformants
Re-plating and purification of plasmid 16 recombinant positive transformants (16-12)
Microscopy examination of plasmid 16 positive transformants
TMA activation of plasmid 16 positive transformants and microscopy verification of GPCR response functionality
Recovery of colorectal cancer epithelial cells (from cryopreserved stocks)
Recovery plating of Saccharomyces boulardii glycerol stocks

Saccharomyces boulardii was cultured under anaerobic conditions in a special medium (simulated intestinal environment). Determination of growth curves.
Quantitative detection of SynAb yeast adhesion to colon cancer epithelial cells.
Experiments in which TMA activates 16 positive transformants and EGFP fluorescence expression intensity is examined by flow cytometry were performed.