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      A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species.

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          Abstract

          Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

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          Author and article information

          Journal
          Proc. Natl. Acad. Sci. U.S.A.
          Proceedings of the National Academy of Sciences of the United States of America
          1091-6490
          0027-8424
          Mar 1 2016
          : 113
          : 9
          Affiliations
          [1 ] Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary;
          [2 ] Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary; csorgo.balint@brc.mta.hu pal.csaba@brc.mta.hu.
          [3 ] Symbiosis and Functional Genomics Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary; SeqOmics Biotechnology Ltd., Mórahalom H-6782, Hungary.
          [4 ] SeqOmics Biotechnology Ltd., Mórahalom H-6782, Hungary.
          Article
          1520040113
          10.1073/pnas.1520040113
          4780621
          26884157

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