pET expression vector customized for efficient seamless cloning

Dragana Dobrijevic; Lily A Nematollahi; Helen C Hailes; John M Ward

doi:10.2144/btn-2020-0101

pET expression vector customized for efficient seamless cloning

Biotechniques. 2020 Nov;69(5):384-387. doi: 10.2144/btn-2020-0101. Epub 2020 Nov 2.

Authors

Dragana Dobrijevic¹, Lily A Nematollahi², Helen C Hailes³, John M Ward¹

Affiliations

¹ Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
² Synthace Ltd, The Westworks, 195 Wood Lane, London, W12 7FQ, UK.
³ Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK.

PMID: 33135501
DOI: 10.2144/btn-2020-0101

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

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacillus subtilis / genetics
Cloning, Molecular / methods*
Genes, Bacterial
Genetic Vectors / metabolism*
Plasmids / genetics*

Abstract

Publication types

MeSH terms

Grants and funding