Part:BBa_K4784002:Experience

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Applications of BBa_K4784002

Experiment
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. Add alanine to the medium. (1) Add 5 µl of 100 mM alanine solution + 995 µl of water + LB liquid medium (just add 25 mg of LB powder) to obtain 500 µM alanine medium. 　(2) 10 µl of 100 mM alanine solution + 990 µl of water + LB liquid medium to obtain 1 mM alanine medium. 　(3) 20 µl of 100 mM alanine solution + 980 µl of water + LB liquid medium to obtain 2 mM alanine medium. 5. 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 

6. Measure the cocentration of Pyruvate every hour

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

ResultExperimental Results Regardless of the concentration of alanine and incubation time, no changes in the concentration and absorbance of pyruvic acid were observed. This suggests that the amino acid transamination reaction may not be proceeding as expected, or that the changes are at a level undetectable within the range of our measurements.

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