Experiments

Molecular Cloning

Molecular cloning was applied to the construction of all plasmids in our project.

This section includes:

1. Plasmid Extraction
2. Agarose Gel Electrophoresis
3. Gel Extraction
4. Heat-Shock Transformation
5. Error-Prone PCR(Beyotime QuickMutation)
6. Gibson Assembly
7. Gateway Assembly

Protein Expression

In the enzyme functional validation experiment, we need to use IPTG to induce protein expression and lyse bacteria to extract the target protein. In terms of protein purification methods, we combined two affinity chromatography purification methods, AKTA Purifier and Ni NTA Sefinose Resin (Pre Packaged Gravity Column), to purify the target protein with His Tag.

This section includes:

1. TB Medium Preparation
2. IPTG Concentrate Preparation
3. Bacteria Culture Method
4. IPTG Induction Protocol
5. Protein Lysis Protocol(For Ni-Resin affinity chromatography gravity column method)
6. Protein Purification Protocol(For Ni-Resin affinity chromatography gravity column method)
7. Protein Staining Protocol(Coomassie brilliant blue R-250)
8. Modified Bradford Protein Assay Kit Protocol
9. Protein Purification Protocol(Using AKTA-Purifier)

Tobacco Cultivation

We transiently transformed our target plasmids into specific tobacco leaves to express our target proteins and synthesize the corresponding metabolites, and we tested the synthesis yields of metabolites by LC-MS/MS.

This section includes:

1. Tobacco cultivation
2. Tobacco transient transfection
3. LC-MS/MS