Title: Simple and Rapid Site-Specific Integration of Multiple Heterologous DNAs into the Escherichia coli Chromosome

Abstract

Escherichia coli is the most studied and well understood microorganism, but research in this system can still be limited by available genetic tools, including the ability to rapidly integrate multiple DNA constructs efficiently into the chromosome. Site-specific, large serine-recombinases can be useful tools, catalyzing a single, unidirectional recombination event between 2 specific DNA sequences, attB and attP, without requiring host proteins for functionality. Using these recombinases, we have developed a system to integrate up to 12 genetic constructs sequentially and stably into in the E. coli chromosome. A cassette of attB sites was inserted into the chromosome and the corresponding recombinases were cloned onto temperature sensitive plasmids to mediate recombination between a non-replicating, attP-containing “cargo” plasmid and the corresponding attB site on the chromosome. The efficiency of DNA insertion into the E. coli chromosome was approximately 10⁷ CFU/μg DNA for six of the recombinases when the competent cells already contained the recombinase-expressing plasmid and approximately 10⁵ CFU/μg DNA or higher when the recombinase-expressing plasmid and “cargo” plasmid were co-transformed. The “cargo” plasmid contains ΦC31 recombination sites flanking the antibiotic gene, allowing for resistance markers to be removed and reused following transient expression of the ΦC31 recombinase. As an example ofmore » the utility of this system, eight DNA methyltransferases from Clostridium clariflavum 4-2a were inserted into the E. coli chromosome to methylate plasmid DNA for evasion of the C. clariflavum restriction systems, enabling the first demonstration of transformation of this cellulose-degrading species.« less

Authors:

Riley, Lauren A. ^[1]; Payne, Irenee C. ^[2]; Tumen-Velasquez, Melissa ^[2]; Guss, Adam M.  ^[1];

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O'Toole, ed., George

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+ Show Author Affiliations

1. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, Bredesen Center, University of Tennessee, Knoxville, Tennessee, USA
2. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA

Publication Date:

Wed Feb 22 00:00:00 EST 2023

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

OSTI Identifier:

1909951

Alternate Identifier(s):

OSTI ID: 1997798

Grant/Contract Number:  

Center for Bioenergy Innovation; SC0019401; AC05-00OR22725; AC02-05CH11231

Resource Type:

Published Article

Journal Name:

Journal of Bacteriology

Additional Journal Information:

Journal Name: Journal of Bacteriology Journal Volume: 205 Journal Issue: 2; Journal ID: ISSN 0021-9193

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; site-specific recombination; phage integrase; genome editing