We used the promoter, RBS, and terminator structures of BBa_K4206001 as is, changing poxB to Pdr12 and attA to AlaDH.And incorporated by Gibson Assembly into pTWV228.

The strong Anderson promoter J23100 was incorporated into the design.
As shown from the modeling, since it is expected to be left for a long period of time in practical use, the pyruvate production would be higher if it is adjusted to keep the pyruvate concentration high. Therefore, we incorporated it into E. coli K12 strain as a constitutive promoter without IPTG induction to increase the amount of said AlaDH.