High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

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Abstract

Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase–assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker–free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

Abstract

Bacteriophage-derived tools enable simple, host-independent construction of complex genetically modified bacterial libraries.

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Most cited references 80

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A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

Nathan Shaner, Gerard Lambert, Andrew Chammas … (2013)

Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.

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A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Carola Engler, Romy Kandzia, Sylvestre Marillonnet (2008)

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.

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Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.

Yu-Xi Jiang, Biao Chen, Chunlan Duan … (2015)

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.

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Author and article information

Contributors

Joshua R. Elmore:

ORCID: https://orcid.org/0000-0003-4750-9640

Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing - original draftRole: Writing - review & editing

Gara N. Dexter:

ORCID: https://orcid.org/0000-0002-1690-5723

Role: ConceptualizationRole: InvestigationRole: Methodology

Henri Baldino:

ORCID: https://orcid.org/0000-0001-7159-3075

Role: Formal analysisRole: InvestigationRole: ResourcesRole: Validation

Jay D. Huenemann:

ORCID: https://orcid.org/0000-0003-0776-8664

Role: Formal analysisRole: Investigation

Ryan Francis:

ORCID: https://orcid.org/0000-0002-3964-6090

Role: InvestigationRole: Validation

George L. Peabody V:

ORCID: https://orcid.org/0000-0001-6761-8575

Role: InvestigationRole: Resources

Jessica Martinez-Baird:

ORCID: https://orcid.org/0000-0003-4656-7793

Role: Validation

Lauren A. Riley: Role: ConceptualizationRole: MethodologyRole: Writing - review & editing

Tuesday Simmons: Role: Methodology

Devin Coleman-Derr:

ORCID: https://orcid.org/0000-0002-6282-1731

Role: ConceptualizationRole: InvestigationRole: ResourcesRole: Writing - review & editing

Adam M. Guss:

ORCID: https://orcid.org/0000-0001-5823-5329

Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing - original draftRole: Writing - review & editing

Robert G. Egbert:

ORCID: https://orcid.org/0000-0002-9470-7124

Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: Writing - original draftRole: Writing - review & editing

Journal

Journal ID (nlm-ta): Sci Adv

Journal ID (iso-abbrev): Sci Adv

Journal ID (publisher-id): sciadv

Journal ID (hwp): advances

Title: Science Advances

Publisher: American Association for the Advancement of Science

ISSN (Electronic): 2375-2548

Publication date Collection: March 2023

Publication date (Electronic, pub): 10 March 2023

Volume: 9

Issue: 10

Electronic Location Identifier: eade1285

Affiliations

[ ¹ ]Biological Science Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.

[ ² ]Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN 37831, USA.

[ ³ ]Bredesen Center for Interdisciplinary Research, University of Tennessee, Knoxville, TN 37996,USA.

[ ⁴ ]Plant and Microbial Biology Department, University of California, Berkeley, CA 94701, USA.

[ ⁵ ]Plant Gene Expression Center, USDA-ARS, Albany, CA 94710, USA.

Author notes

[* ]Corresponding author. Email: robert.egbert@ 123456pnnl.gov (R.G.E.); gussam@ 123456ornl.gov (A.M.G.).

Author information

Joshua R. Elmore https://orcid.org/0000-0003-4750-9640

Gara N. Dexter https://orcid.org/0000-0002-1690-5723

Henri Baldino https://orcid.org/0000-0001-7159-3075

Jay D. Huenemann https://orcid.org/0000-0003-0776-8664

Ryan Francis https://orcid.org/0000-0002-3964-6090

George L. Peabody V https://orcid.org/0000-0001-6761-8575

Jessica Martinez-Baird https://orcid.org/0000-0003-4656-7793

Devin Coleman-Derr https://orcid.org/0000-0002-6282-1731

Adam M. Guss https://orcid.org/0000-0001-5823-5329

Robert G. Egbert https://orcid.org/0000-0002-9470-7124

Article

Publisher ID: ade1285

DOI: 10.1126/sciadv.ade1285

PMC ID: 10005180

PubMed ID: 36897939

SO-VID: 54b07461-9cd9-4ff6-999e-257d8e3b98b8

Copyright © Copyright © 2023 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

History

Date received : 15 August 2022

Date accepted : 01 February 2023

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000015, U.S. Department of Energy;

Award ID: Persistence Control Science Focus Area

Funded by: FundRef http://dx.doi.org/10.13039/100000015, U.S. Department of Energy;

Award ID: Agile BioFoundry

Funded by: FundRef http://dx.doi.org/10.13039/100000015, U.S. Department of Energy;

Award ID: Center for Bioenergy Innovation

Funded by: FundRef http://dx.doi.org/10.13039/100000185, Defense Advanced Research Projects Agency;

Award ID: HR0011045664

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Copyeditor Nicole Falcasantos

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High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

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BIO Integration

Most cited references 80

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.

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Cited by 17

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