Figure 4.

Construction and expression of the linear expression templates by PCR. (A) Schematic diagram of the PCR-based generation of the ompA fusion constructs. Three primary PCR products with defined overlapping ends were synthesized by the first PCR. These three fragments were joined in the second PCR, overlap extension PCR, which also simultaneously introduced the regulatory elements and the OmpA signal peptide sequence to the target genes. The red bar indicates the overlapping region. The blue and black bar indicate UTR and ORF, respectively. (B) Agarose gel electrophoresis of the PCR products. Second PCR products have an additional 600 bp as a result of the fusion with 5′-UTR and 3′-UTR. (C) Cell-free expression of ompA-fused PCR products. The reaction mixture for cell-free protein synthesis was prepared as described in Materials and Methods. After 2 h of incubation, 2 μl of the reaction mixture was loaded on a 13% Tricine-SDS–PAGE gel and stained with Coomassie Brilliant Blue. Cell-free expressed proteins are indicated by arrows.