Fig 2.
Verification of different types of engineered bacteria during the generation of the afeI deletion and overexpression strains. A. Diagram of the afeI gene cluster and verification primers. UHA and DHA represent upstream and downstream homologous arms respectively. B. Electrophoretic analysis of PCR products to verify ΔafeI. Lanes 1, 3 and 5, PCR products from ΔafeI using the primer pair P1F/R, P2F/R and P3F/R respectively; lanes 2, 4 and 6, PCR products from wild type using the primer pair P1F/R, P2F/R and P3F/R respectively. C. Illustration of the afeI expression vector pJRD215‐Ptac‐afeI and verification primers. D. Electrophoretic analysis of PCR products to verify the afeI‐overexpression strains. Lanes 1, 2, 3 and 4, PCR products from the afeI overexpression strain, the wild‐type strain, pJRD215 and pJRD215‐Ptac‐afeI respectively, using the primer pair P4F/R. E. Diagram of the act gene cluster and verification primers. F. Electrophoretic analysis of PCR products to verify Δact(afeI). Lanes 1, 3 and 5, PCR products from Δact(afeI) using the primer pair PA1F/R, PA2F/R and PA3F/R respectively; lanes 2, 4 and 6, PCR products from wild type using the primer pair PA1F/R, PA2F/R and PA3F/R respectively; lanes 7, 8, 9 and 10, PCR products from the Δact(afeI) strain, the wild‐type strain, pJRD215 and pJRD215‐Ptac‐afeI respectively, using the primer pair P4F/R. G. Diagram of the ΔafeI&act gene cluster and verification primers. H. Electrophoretic analysis of PCR products to verify ΔafeI&act. Lanes 1, 3, 5, 7, 9 and 11, PCR products from ΔafeI&act using the primer pair P1F/R, P2F/R, P3F/R, PA1F/R, PA2F/R and PA3F/R respectively; lanes 2, 4, 6, 8, 10 and 12, PCR products from wild type using the primer pair P1F/R, P2F/R, P3F/R, PA1F/R, PA2F/R and PA3F/R respectively. [Color figure can be viewed at wileyonlinelibrary.com]