Journal: Applied and Environmental Microbiology
Article Title: Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain ▿
Figure Lengend Snippet: PCR analysis of three disruption mutants. P1 to P8 refer to priming sites. PCR amplification for identifying the deletion of each target gene was performed using each E. coli BL21 mutant genomic DNA as a template. (A) Salvage pathway gene disruptions. M, size marker; lane 1, deoC and deoB region including deoA (2.0 kb); lane 2, Δ deoC :: cat ::Δ deoB (1.3 kb); lane 3, Δ( deoC-deoB ) (0.4 kb), represented to Δ deoA ; lane 4, tdk (0.6 kb); lane 5, Δ tdk :: cat (1.1 kb); lane 6, Δ tdk (0.2 kb); lane 7, udp (0.7 kb); lane 8, Δ udp :: cat (1.1 kb); lane 9, Δ udp (0.2 kb). The FRT (black bar)-flanked chloramphenicol-resistant gene was amplified by PCR. The linear disruption PCR fragment (gray bar) was transformed into a strain expressing the λ Red recombinase, and then chloramphenicol-resistant transformants were selected. The selective marker was eliminated by FLP recombinase system. (B) ung disruption. Lane 1, ung (0.7 kb); lane 2, Δ ung :: cat (1.1 kb); lane 3, Δ ung (0.2 kb).
Article Snippet: PCR mixtures containing 1 U thermostable DNA polymerase (Roche Molecular Biomedicals, Basel, Switzerland), 20 mM Tris (pH 8.4), 1.5 mM MgCl2 , 1 μM gene disruption primers, 0.2 mM deoxynucleoside triphosphates, and 0.5 μg ml−1 of plasmid pKD3 were incubated at 94°C for 5 min, followed by 30 cycles at 94°C (45 s), 55°C (45 s), and 72°C (1 min), and followed by a final extension time of 10 min at 72°C.
Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis, Marker, Transformation Assay, Expressing