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Title: Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cell in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants maymore » be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less
Authors:
 [1] ;  [2] ;  [3] ;  [2] ;  [2] ;  [4] ;  [4] ;  [2] ;  [5]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Agile BioFoundry, Emeryville, CA (United States
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Agile BioFoundry, Emeryville, CA (United States
  5. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering and Dept. of Chemical and Biomolecular Engineering; Technical Univ. of Denmark, Lyngby (Denmark). The Novo Nordisk Foundation Center for Biosustainability
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
SC-23.2 USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23). Biological Systems Science Division; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B); Univ. of California, Berkeley, CA (United States); Rose Hills Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Applied microbiology; Mutation
OSTI Identifier:
1425440

Thompson, Mitchell G., Sedaghatian, Nima, Barajas, Jesus F., Wehrs, Maren, Bailey, Constance B., Kaplan, Nurgul, Hillson, Nathan J., Mukhopadhyay, Aindrila, and Keasling, Jay D.. Isolation and characterization of novel mutations in the pSC101 origin that increase copy number. United States: N. p., Web. doi:10.1038/s41598-018-20016-w.
Thompson, Mitchell G., Sedaghatian, Nima, Barajas, Jesus F., Wehrs, Maren, Bailey, Constance B., Kaplan, Nurgul, Hillson, Nathan J., Mukhopadhyay, Aindrila, & Keasling, Jay D.. Isolation and characterization of novel mutations in the pSC101 origin that increase copy number. United States. doi:10.1038/s41598-018-20016-w.
Thompson, Mitchell G., Sedaghatian, Nima, Barajas, Jesus F., Wehrs, Maren, Bailey, Constance B., Kaplan, Nurgul, Hillson, Nathan J., Mukhopadhyay, Aindrila, and Keasling, Jay D.. 2018. "Isolation and characterization of novel mutations in the pSC101 origin that increase copy number". United States. doi:10.1038/s41598-018-20016-w. https://www.osti.gov/servlets/purl/1425440.
@article{osti_1425440,
title = {Isolation and characterization of novel mutations in the pSC101 origin that increase copy number},
author = {Thompson, Mitchell G. and Sedaghatian, Nima and Barajas, Jesus F. and Wehrs, Maren and Bailey, Constance B. and Kaplan, Nurgul and Hillson, Nathan J. and Mukhopadhyay, Aindrila and Keasling, Jay D.},
abstractNote = {pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cell in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.},
doi = {10.1038/s41598-018-20016-w},
journal = {Scientific Reports},
number = 1,
volume = 8,
place = {United States},
year = {2018},
month = {1}
}

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