Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering

Bubnov, D. M.; Yuzbashev, Tigran V.; Vybornaya, Tatiana V.; Нетрусов, А.И.; Sineoky, S. P.

doi:10.1016/j.mimet.2018.06.001

Cited by 11 publications

(7 citation statements)

References 37 publications

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“…Plasmids pRedCm, pRedCmOcr or the chromosomally integrated ocr-γ βexo-P L -cat module were employed for expressing the Red recombination functions. The plasmid pDL17 (26) was used as a helper for integrating cassettes that confer chloramphenicol resistance. A detailed description for preparing electrocompetent cells and linear DNA cassettes is presented under 'Preparation of linear cassettes' and 'Preparation of electrocompetent cells' in the Supplementary Material and Methods.…”

Section: Red Recombineeringmentioning

confidence: 99%

“…Therefore, we cloned this transcription unit into a helper plasmid expressing Red functions under control of the P rhaB promoter, thereby producing the pRedCm helper plasmid. Remarkably, this plasmid is based on the previously described pMB1-derived conditional origin of replication (26), whose activity is controlled by the LacIrepressible P T5/lacO promoter and is severely inhibited by IPTG supplementation. The pRedCm plasmid could be easily eliminated from host cells by two rounds of re-streaking as described under 'Helper plasmid curing' in the Supplementary Material and Methods.…”

Section: Design Of a Counterselection Strategymentioning

confidence: 99%

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Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs

Bubnov

Yuzbashev

Khozov

et al. 2022

Nucleic Acids Research

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Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50–100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

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Section: Red Recombineeringmentioning

confidence: 99%

Section: Design Of a Counterselection Strategymentioning

confidence: 99%

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs

Bubnov

Yuzbashev

Khozov

et al. 2022

Nucleic Acids Research

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“…Recently, the use of dominant-negative mutator alleles of conserved DNA MMR proteins (e.g., MutS and MutL) was implemented in genome engineering. Importantly, controlled expression of a mutator allele aided the transient inhibition of MMR in trans only during the recombination process and allows recombineering and efficient OMAR in wild type cells (Nyerges et al, 2016;Bubnov et al, 2018).…”

Section: Oligo-mediated Allelic-replacement (Omar) Avoids the Use Of mentioning

confidence: 99%

Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics

2020

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Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering-or recombinationmediated genetic engineering-represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ∼40-50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double-and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls.

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“…reported the construction of a plasmid system with a dominant-negative mutS allele and a novel conditionally replicating origin that enabled plasmid propagation and genome editing at higher temperatures (e.g. 37°C) (32). Finally, Lennen et al.…”

Section: Introductionmentioning

confidence: 99%

A versatile platform strain for high-fidelity multiplex genome editing

Egbert

Rishi

Adler

et al. 2019

Nucleic Acids Research

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Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

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Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering

Cited by 11 publications

References 37 publications

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs

Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics

A versatile platform strain for high-fidelity multiplex genome editing

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