Experiments

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