Abstract
pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used modern methods of DNA assembly and directed evolution to identify improved designs for these modules and demonstrated that these designs support higher protein production yields. Herein, we present two PCR protocols for implementing the designs and increasing protein production from existing pET expression plasmids. Graphic abstract: A simple workflow for implementing novel designs in pET expression plasmids.