Preliminary idea determination
Search and screening of cholesterol degrading genes
Search for a suitable promoter: oleic acid inducer was finally identified
The design of gene combination

Gene synthesis and plasmid construction

Prepared the competent
Bacterial culture and plasmid extraction
Transformed the target gene into the chassis organism Escherichia coli Nissel 1917

Functional verification of IsmA and BCoAT genes, including:
Cholesterol was measured by phthalaldehyde
SCFA production was measured by gas chromatography
The bacteria were lysed by sonication for BSH crude enzyme solution extraction

Qualitative detection of bile saline hydrolase
Prepared egg yolk medium and perform the detection of cholesterol degradation from food sources
Performed cell counting to detect bacterial growth status

Verified the function of oleic acid inducer by microplate reader and mRFP
Confirmed the expression of IsmA and BSH by SDS-PAGE
Determined the downstream products of IsmA conversion to cholesterol by UHPLC