Title: 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 » be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Authors:

Thompson, Mitchell G. ^[1]; Sedaghatian, Nima ^[2]; Barajas, Jesus F. ^[3]; Wehrs, Maren ^[2]; Bailey, Constance B. ^[2]; Kaplan, Nurgul ^[4]; Hillson, Nathan J. ^[4]; Mukhopadhyay, Aindrila ^[2]; Keasling, Jay D. ^[5]

---

+ Show Author Affiliations

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:

Thu Jan 25 00:00:00 EST 2018

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