Fig. 4.

Sequence repeat assembly through the PS-Brick scheme. a Construction of CRISPR array containing sequence repeats via TA clone/BciVI based PS-Brick assembly. The N20 sequence flanked with the same promoter pJ23119 and the sgRNA sequence located in the pTargetET vector. The entrance site of HindIII/BciVI and editing template was introduced into pTargetF to generate original vector ptargetET for PS-Brick assembly. The N20 sequences of tdh and ilvA fixed with the same promoter and sgRNA at each end were sequentially inserted into ptargetET at twice PS-Brick reaction. b Identification of sgRNA arrays constructed into the pTarget vector. c Identification of tdh and ilvA gene deletion. d Fed-batch fermentation of the engineering strain in 7.5 L fermentor. Strain MG1655△tdh△ilvA/pACYC184-thrA⁴³³BC-asd-P_TBCD1-rhtC (solid symbol), strain MG1655/pACYC184-thrA⁴³³BC-asd-P_TBCD1-rhtC The control strain (open symbol), biomass (triangle), threonine (circle) and isoleucine (square). Data shown are mean values from three replicates, and the standard deviations are presented