1. Gibson assembly

Forming the plasmid contains Pdr12 and AlaDH.

2. Transformation

Following the Single Tube Transformation Protocol provided by iGEM, except for the process of heat shock.
We conducted heat-shock for 30sec at 42°C on a heat block.

3. Culturing

Pick colonies and culture them in LB-cam containing ampicillin overnight.

4. IPTG induction

Add 30μl of culture fluid to 3 ml of LB-medium containing ampicillin and 0-1.5μmol of IPTG.
Cultured for 4-5h at 37℃ in culture tubes in a shaking water bath at 168 min^-1

5. Measure the cocentration of Pyruvate every hour

* According to the protocol offered by assay kit's manifacture, make the standard curve.
The protocols is here; https://docs.aatbio.com/products/protocol-and-product-information-sheet-pis/protocol-for-amplite-colorimetric-pyruvate-assay-kit-version-5766863da6.pdf