1. Preparation of LB Medium
2. Incubate strains containing plasmids

Pick the mono-colony and incubate in LB liquid culture medium

1. Plasmids extraction
2. PCR amplification for lipase
3. Double enzyme Digestion and gel extraction
4. T4 DNA ligase to construct the plasmids
5. Transform the plasmids into DH5α competent cells and coat them on the solid LB medium plates

1. mono-colony identification and inoculate into liquid LB culture medium
2. agarose gel electrophoresis

1. Plasmid extraction and double-enzyme digestion
2. gel electrophoresis and send for Sanger sequencing

Transform the correct plasmids into BL21(DE3) competent cells and coat on the solid LB medium plate

Inoculate mono-colony into liquid LB medium, 37℃, 200rpm overnight

1. Transfer the overnight cultured medium into 1L LB liquid medium
2. Add IPTG to induce protein expression when OD600 is arranging 0.4-0.6, and culture at 37℃, 170rpm

1. SDS-PAGE electrophoresis solution preparation
2. Protein purification

Measure the standard curve of enzyme activity

Detection of lipase activity at different pH values

Detection of lipase activity assay at different temperatures