ArticleNucleic acids research2014
Rational optimization of tolC as a powerful dual selectable marker for genome engineering.
Article in Nucleic acids research, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
26 citing papers in PubMed, 38 citations in OpenAlex.
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- Efficient Bacterial Genome Engineering throughout the Central Dogma Using the Dual-Selection MarkerACS synthetic biology · 2022Article
- Past, Present, and Future of Genome Modification inMicroorganisms · 2022Review
- Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.Nucleic acids research · 2022Article
- Characterization of ColE1 Production for RobustACS synthetic biology · 2022Article
- An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.Plants (Basel, Switzerland) · 2022Article
- Genetic Manipulation of Wild Human GutJournal of bacteriology · 2020Article
- Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes.Cell chemical biology · 2018Article
- Utilization of rare codon-rich markers for screening amino acid overproducers.Nature communications · 2018Article
- Codon usage of highly expressed genes affects proteome-wide translation efficiency.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Chromosomal barcoding as a tool for multiplexed phenotypic characterization of laboratory evolved lineages.Scientific reports · 2018Article
- A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.Microbial cell factories · 2017Article
- Optimizing complex phenotypes through model-guided multiplex genome engineering.Genome biology · 2017Article
- Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
- Engineering microbial hosts for production of bacterial natural products.Natural product reports · 2016Review
- PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli.PloS one · 2016Article
- Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.Nature protocols · 2015Article
- Genomes by design.Nature reviews. Genetics · 2015Review
Corrections and comments
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
Abstract
Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.