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Cited by 3CRISPR and Genetic EngineeringMicrobial Metabolic Engineering and BioproductionBacterial Genetics and Biotechnology

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Strategies to identify and edit improvements in synthetic genome segments episomally

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Rudolph, Alexandra, Nyerges, Akos, Chiappino-Pepe, Anush, Landon, Matthieu, Baas-Thomas, Maximilien, Church, George·2023·Nucleic Acids Research
DOI 10.1093/nar/gkad692PMC PMC10570025

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Abstract

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to recoding an Escherichia coli strain to replace seven codons with synonymous alternatives genome wide. Ten 44 402–47 179 bp de novo synthesized DNA segments contained in a BAC from the recoded strain were unable to complement deletion of the corresponding 33–61 wild-type genes using a single antibiotic resistance marker. Next-generation sequencing (NGS) was used to identify 1–7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the recoded segment contained in the BAC when both the recoded and wild-type copies of the mutated genes had to exist by necessity during the repair process. Finally, two web-based tools were used to predict the impact of a subset of non-recoding missense mutations on strain fitness using protein structure and function calls.

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Metadata

Authors
Rudolph, A., Nyerges, A., Chiappino-Pepe, A., Landon, M., Baas-Thomas, M., & Church, G.
Affiliations
  • Harvard University (US)
  • Department of Genetics, Harvard Medical School , Boston , MA  02115 , USA
Corresponding author
Rudolph, Alexandra
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2023-08-24
Volume
51
Issue
18
Pages
10094-10106
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkad692
PMC
PMC10570025
OpenAlex
W4386116000
Cited by
3
References
6
Citations by year
2025: 1 · 2024: 2
FWCI
0.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
Funders
The United States Department of Energy (DE-FG02-02ER63445)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
recombineering, bacterial artificial chromosome, synthetic genome, genome editing, Escherichia coli, missense mutations, strain fitness, de novo DNA synthesis
MeSH terms
Gene Editing, Escherichia coli, Genetic Engineering, Mutation, Plasmids, Genome, Bacterial, Genes, Essential, Chromosomes, Artificial, Bacterial, High-Throughput Nucleotide Sequencing

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