Engineering an Escherichia coli strain for production of long single-stranded DNA

Shen, Konlin; Flood, Jake J.; Zhang, Zhihuizi; Ha, Alvin; Shy, Brian R.; Dueber, John E.; Douglas, Shawn M.

doi:10.1093/nar/gkae189

Title: Engineering an Escherichia coli strain for production of long single-stranded DNA

Journal Article · 17 March 2024 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkae189 · OSTI ID:2470714

Shen, Konlin ^[1]; Flood, Jake J. ^[2]; Zhang, Zhihuizi ^[1]; Ha, Alvin ^[3]; Shy, Brian R. ^[4]; Dueber, John E. ^[5];

^[1]

University of California, San Francisco, CA (United States)
University of California, Berkeley, CA (United States)
University of California, San Francisco, CA (United States); Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA (United States)
University of California, San Francisco, CA (United States); Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA (United States)
University of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli ‘helper’ strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470714

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 7 Vol. 52; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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