Fig. 10

Schematic representation of vector construction. The base vector pPPD was cut with corresponding restriction site to insert ORF1 (single constructs). The addition of next ORFs was performed by cutting the new vector with BcuI and PstI (or BamHI) and the next insert with XbaI and PstI (or BamHI) and creation a BcuI/XbaI scar. DAHP synthase encoded by aroG was amplified from E. coli using E. coli cells as template. The gene was inserted into the integrative vector pEERM3. The expression was driven by a constitutive promotor Ptrc2O and synthetic ribosomal binding site RBS*, the gene sequence was followed by a flexible Gly-Ser linker and Strep-tag sequence at the C-terminal position. In order to create a feedback-resistant version of E.coli DAHP synthase, a Pro150Leu substitution [5] was introduced by mutagenesis PCR, creating the construct AroG150-pEERM3