pET expression vector customized for efficient seamless cloning

Abstract

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.

Keywords

• expression in E. coli
• Golden Gate
• pET vectors
• sacB
• seamless cloning