Isolation and characterization of novel mutations in the pSC101 origin that increase copy number
Abstract
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 »
- Authors:
-
- 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
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Agile BioFoundry, Emeryville, CA (United States
- 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
- 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:
- Research Org.:
- Lawrence Berkeley National Laboratory (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), Sustainable Transportation Office. Bioenergy Technologies Office; Univ. of California, Berkeley, CA (United States); Rose Hills Foundation
- OSTI Identifier:
- 1425440
- Grant/Contract Number:
- AC02-05CH11231
- Resource 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
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Applied microbiology; Mutation
Citation Formats
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., 2018.
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. https://doi.org/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. Thu .
"Isolation and characterization of novel mutations in the pSC101 origin that increase copy number". United States. https://doi.org/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 = {Thu Jan 25 00:00:00 EST 2018},
month = {Thu Jan 25 00:00:00 EST 2018}
}
Web of Science
Works referenced in this record:
Negative control of plasmid pSC101 replication by increased concentrations of both initiator protein and iterons.
journal, January 2000
- Furuno, Sigekazu; Watanabe-Murakami, Yuhko; Takebe-Suzuki, Naoko
- The Journal of General and Applied Microbiology, Vol. 46, Issue 1
Characterization of copy number mutants of plasmid pSC101.
journal, January 1997
- Wadood, Abdul; Dohmoto, Mitsuko; Sugiura, Shigeki
- The Journal of General and Applied Microbiology, Vol. 43, Issue 6
Twenty years of the pPS10 replicon: insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids
journal, September 2004
- Giraldo, Rafael; Fernández-Tresguerres, M. Elena
- Plasmid, Vol. 52, Issue 2
Pseudomonas putida—a versatile host for the production of natural products
journal, June 2015
- Loeschcke, Anita; Thies, Stephan
- Applied Microbiology and Biotechnology, Vol. 99, Issue 15
mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system
journal, September 1998
- Carrier, Trent; Jones, Kristala L.; Keasling, J. D.
- Biotechnology and Bioengineering, Vol. 59, Issue 6
Factors influencing gene expression and resistance for Gram-negative organisms expressing plasmid-encoded ampC genes of Enterobacter origin
journal, April 2003
- Reisbig, M. D.
- Journal of Antimicrobial Chemotherapy, Vol. 51, Issue 5
Control of plasmid DNA replication by iterons: no longer paradoxical: Control of DNA replication by iterons
journal, August 2000
- Chattoraj, Dhruba K.
- Molecular Microbiology, Vol. 37, Issue 3
I-TASSER: a unified platform for automated protein structure and function prediction
journal, March 2010
- Roy, Ambrish; Kucukural, Alper; Zhang, Yang
- Nature Protocols, Vol. 5, Issue 4
New pSC101-derivative cloning vectors with elevated copy numbers
journal, May 2008
- Peterson, James; Phillips, Gregory J.
- Plasmid, Vol. 59, Issue 3
A noncytotoxic DsRed variant for whole-cell labeling
journal, October 2008
- Strack, Rita L.; Strongin, Daniel E.; Bhattacharyya, Dibyendu
- Nature Methods, Vol. 5, Issue 11
j5 DNA Assembly Design Automation Software
journal, December 2011
- Hillson, Nathan J.; Rosengarten, Rafael D.; Keasling, Jay D.
- ACS Synthetic Biology, Vol. 1, Issue 1, p. 14-21
Correlation analysis of targeted proteins and metabolites to assess and engineer microbial isopentenol production: Targeted Proteomics-Based Correlation Analysis
journal, May 2014
- George, Kevin W.; Chen, Amy; Jain, Aakriti
- Biotechnology and Bioengineering, Vol. 111, Issue 8
Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production
journal, September 2013
- Alonso-Gutierrez, Jorge; Chan, Rossana; Batth, Tanveer S.
- Metabolic Engineering, Vol. 19
BglBrick vectors and datasheets: A synthetic biology platform for gene expression
journal, January 2011
- Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong
- Journal of Biological Engineering, Vol. 5, Issue 1
The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data
journal, July 2010
- McKenna, A.; Hanna, M.; Banks, E.
- Genome Research, Vol. 20, Issue 9
The Acinetobacter sp. chnB promoter together with its cognate positive regulator ChnR is an attractive new candidate for metabolic engineering applications in bacteria
journal, March 2008
- Steigedal, Magnus; Valla, Svein
- Metabolic Engineering, Vol. 10, Issue 2
Transcription Factor-Based Screens and Synthetic Selections for Microbial Small-Molecule Biosynthesis
journal, September 2012
- Dietrich, Jeffrey A.; Shis, David L.; Alikhani, Azadeh
- ACS Synthetic Biology, Vol. 2, Issue 1
Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).
journal, April 1994
- Ishiai, M.; Wada, C.; Kawasaki, Y.
- Proceedings of the National Academy of Sciences, Vol. 91, Issue 9
DeviceEditor visual biological CAD canvas
journal, December 2012
- Chen, Joanna; Densmore, Douglas; Ham, Timothy S.
- Journal of Biological Engineering, Vol. 6, Issue 1
Structural basis for regulation of bifunctional roles in replication initiator protein
journal, November 2007
- Nakamura, A.; Wada, C.; Miki, K.
- Proceedings of the National Academy of Sciences, Vol. 104, Issue 47
Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli
journal, March 2007
- Pitera, Douglas J.; Paddon, Chris J.; Newman, Jack D.
- Metabolic Engineering, Vol. 9, Issue 2
Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin.
journal, September 1991
- Wickner, S.; Hoskins, J.; McKenney, K.
- Proceedings of the National Academy of Sciences, Vol. 88, Issue 18
Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools
journal, June 2012
- Ham, T. S.; Dmytriv, Z.; Plahar, H.
- Nucleic Acids Research, Vol. 40, Issue 18
Metabolic engineering of Escherichia coli for the production of four-, five- and six-carbon lactams
journal, May 2017
- Chae, Tong Un; Ko, Yoo-Sung; Hwang, Kyu-Sang
- Metabolic Engineering, Vol. 41
Development of a Transcription Factor-Based Lactam Biosensor
journal, November 2016
- Zhang, Jingwei; Barajas, Jesus F.; Burdu, Mehmet
- ACS Synthetic Biology, Vol. 6, Issue 3
I-TASSER server for protein 3D structure prediction
journal, January 2008
- Zhang, Yang
- BMC Bioinformatics, Vol. 9, Issue 1
UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004
- Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
- Journal of Computational Chemistry, Vol. 25, Issue 13
An Easy and Accurate Agarose Gel Assay for Quantitation of Bacterial Plasmid Copy Numbers
journal, August 2000
- Pushnova, Elena A.; Geier, Michael; Zhu, Yu Sheng
- Analytical Biochemistry, Vol. 284, Issue 1
Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009
- Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
- Nature Methods, Vol. 6, Issue 5, p. 343-345
Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon
journal, April 2002
- Smolke, Christina D.; Keasling, Jay D.
- Biotechnology and Bioengineering, Vol. 78, Issue 4, p. 412-424
Correlation analysis of targeted proteins and metabolites to assess and engineer microbial isopentenol production: Targeted Proteomics-Based Correlation Analysis
journal, May 2014
- George, Kevin W.; Chen, Amy; Jain, Aakriti
- Biotechnology and Bioengineering, Vol. 111, Issue 8
An Easy and Accurate Agarose Gel Assay for Quantitation of Bacterial Plasmid Copy Numbers
journal, August 2000
- Pushnova, Elena A.; Geier, Michael; Zhu, Yu Sheng
- Analytical Biochemistry, Vol. 284, Issue 1
Pseudomonas putida—a versatile host for the production of natural products
journal, June 2015
- Loeschcke, Anita; Thies, Stephan
- Applied Microbiology and Biotechnology, Vol. 99, Issue 15
Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli
journal, March 2007
- Pitera, Douglas J.; Paddon, Chris J.; Newman, Jack D.
- Metabolic Engineering, Vol. 9, Issue 2
The Acinetobacter sp. chnB promoter together with its cognate positive regulator ChnR is an attractive new candidate for metabolic engineering applications in bacteria
journal, March 2008
- Steigedal, Magnus; Valla, Svein
- Metabolic Engineering, Vol. 10, Issue 2
Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production
journal, September 2013
- Alonso-Gutierrez, Jorge; Chan, Rossana; Batth, Tanveer S.
- Metabolic Engineering, Vol. 19
Metabolic engineering of Escherichia coli for the production of four-, five- and six-carbon lactams
journal, May 2017
- Chae, Tong Un; Ko, Yoo-Sung; Hwang, Kyu-Sang
- Metabolic Engineering, Vol. 41
Transcription Factor-Based Screens and Synthetic Selections for Microbial Small-Molecule Biosynthesis
journal, September 2012
- Dietrich, Jeffrey A.; Shis, David L.; Alikhani, Azadeh
- ACS Synthetic Biology, Vol. 2, Issue 1
A noncytotoxic DsRed variant for whole-cell labeling
journal, October 2008
- Strack, Rita L.; Strongin, Daniel E.; Bhattacharyya, Dibyendu
- Nature Methods, Vol. 5, Issue 11
Machine learning discovery of missing links that mediate alternative branches to plant alkaloids
journal, March 2022
- Vavricka, Christopher J.; Takahashi, Shunsuke; Watanabe, Naoki
- Nature Communications, Vol. 13, Issue 1
Continuous evolution of base editors with expanded target compatibility and improved activity
journal, July 2019
- Thuronyi, Benjamin W.; Koblan, Luke W.; Levy, Jonathan M.
- Nature Biotechnology, Vol. 37, Issue 9
Control of plasmid DNA replication by iterons: no longer paradoxical: Control of DNA replication by iterons
journal, August 2000
- Chattoraj, Dhruba K.
- Molecular Microbiology, Vol. 37, Issue 3
Structural basis for regulation of bifunctional roles in replication initiator protein
journal, November 2007
- Nakamura, A.; Wada, C.; Miki, K.
- Proceedings of the National Academy of Sciences, Vol. 104, Issue 47
Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin.
journal, September 1991
- Wickner, S.; Hoskins, J.; McKenney, K.
- Proceedings of the National Academy of Sciences, Vol. 88, Issue 18
Factors influencing gene expression and resistance for Gram-negative organisms expressing plasmid-encoded ampC genes of Enterobacter origin
journal, April 2003
- Reisbig, M. D.
- Journal of Antimicrobial Chemotherapy, Vol. 51, Issue 5
Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools
journal, June 2012
- Ham, T. S.; Dmytriv, Z.; Plahar, H.
- Nucleic Acids Research, Vol. 40, Issue 18
I-TASSER server for protein 3D structure prediction
journal, January 2008
- Zhang, Yang
- BMC Bioinformatics, Vol. 9, Issue 1
BglBrick vectors and datasheets: A synthetic biology platform for gene expression
journal, January 2011
- Lee, Taek; Krupa, Rachel A.; Zhang, Fuzhong
- Journal of Biological Engineering, Vol. 5, Issue 1
DeviceEditor visual biological CAD canvas
journal, January 2012
- Chen, Joanna; Densmore, Douglas; Ham, Timothy S.
- Journal of Biological Engineering, Vol. 6, Issue 1
Characterization of copy number mutants of plasmid pSC101.
journal, January 1997
- Wadood, Abdul; Dohmoto, Mitsuko; Sugiura, Shigeki
- The Journal of General and Applied Microbiology, Vol. 43, Issue 6
Works referencing / citing this record:
De novo DNA synthesis using polymerase-nucleotide conjugates
journal, June 2018
- Palluk, Sebastian; Arlow, Daniel H.; de Rond, Tristan
- Nature Biotechnology, Vol. 36, Issue 7
Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis
journal, September 2019
- Brenac, Léa; Baidoo, Edward E. K.; Keasling, Jay D.
- Molecular Microbiology, Vol. 112, Issue 5
Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles
journal, January 2019
- Tran, Frances; Boedicker, James Q.
- Journal of Bacteriology, Vol. 201, Issue 7
Fluorescent amplification for next generation sequencing (FA-NGS) library preparation
journal, January 2020
- Chiniquy, Jennifer; Garber, Megan E.; Mukhopadhyay, Aindrila
- BMC Genomics, Vol. 21, Issue 1
Measuring cis-regulatory energetics in living cells using allelic manifolds
journal, December 2018
- Forcier, Talitha L.; Ayaz, Andalus; Gill, Manraj S.
- eLife, Vol. 7
Fluorescent amplification for next generation sequencing (FA-NGS) library preparation
journal, January 2020
- Chiniquy, Jennifer; Garber, Megan E.; Mukhopadhyay, Aindrila
- BMC Genomics, Vol. 21, Issue 1