A Bacterial Expression Vector Archive(BEVA) for flexible modular assembly of Golden Gate-compatible vectors

ABSTRACTWe present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. To demonstrate the potential of the system three example vectors were constructed and tested. Golden Gate level 1 vectors; pOGG024, with a broad-host range and high copy number was used for gene expression in laboratory-culturedRhizobium leguminosarum, and pOGG026, with a broad-host range a lower copy number and excellent stability, even in the absence of antibiotic selection. The application of pOGG026 is demonstrated in environmental samples by bacterial gene expression in nitrogen-fixing nodules on pea plants roots formed byR. leguminosarum. Finally, the level 2 cloning vector pOGG216 is a broad-host range, medium copy number, for which we demonstrate an application by constructing a dual reporter plasmid expressing green and red fluorescent proteins.IMPORTANCEModular assembly is powerful as it allows easy combining of different components from a library of parts. In designing a modular vector assembly system, the key constituent parts (and modules) are; an origin of plasmid replication, antibiotic resistance marker(s), cloning site(s), together with additional accessory modules as required. In an ideal vector, the size of each module would be minimized, and this we have addressed. We have designed such a vector assembly system by utilizing the Type IIS restriction enzyme Esp3I and have demonstrated its use for Golden Gate cloning inEscherichia coli. An important attribute of this modular vector assembly is that using the principles outlined here, new modules for specific applications, e.g. origin of replication for plasmids in other bacteria, can easily be designed. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach.

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Construction of a broad host range expression plasmid vector by Golden Gate cloning

Malaysian Journal of Microbiology ◽

10.21161/mjm.211184 ◽

2021 ◽

Author(s):

Teo, Y. L. ◽

Toh, W. K. ◽

Tor, X. Y. ◽

Ho, C.-L. ◽

Loh, P. C. ◽

...

Keyword(s):

Host Range ◽

Plasmid Vector ◽

Broad Host Range ◽

Golden Gate ◽

Expression Plasmid ◽

Golden Gate Cloning

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Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

Synthetic Biology ◽

10.1093/synbio/ysab003 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Marcos Valenzuela-Ortega ◽

Christopher French

Keyword(s):

Copy Number ◽

Life Science ◽

Insertion Site ◽

Golden Gate ◽

Common Elements ◽

Modern Life ◽

Specific Host ◽

Modular Cloning ◽

Golden Gate Cloning ◽

Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

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Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control

Plasmids in Bacteria ◽

10.1007/978-1-4613-2447-8_16 ◽

1985 ◽

pp. 173-188 ◽

Cited By ~ 3

Author(s):

Richard J. Meyer ◽

Lung-Shen Lin ◽

Kyunghoon Kim ◽

Michael A. Brasch

Keyword(s):

Host Range ◽

Copy Number ◽

Broad Host Range ◽

Copy Number Control ◽

Broad Host Range Plasmid

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Applications of Golden Gate cloning to protein production using the baculovirus expression vector system

10.1016/bs.mie.2021.05.017 ◽

2021 ◽

pp. 155-169

Author(s):

Erich Spielvogel ◽

Jana Neuhold ◽

Peggy Stolt-Bergner

Keyword(s):

Protein Production ◽

Expression Vector ◽

Vector System ◽

Golden Gate ◽

Baculovirus Expression Vector System ◽

Baculovirus Expression ◽

Baculovirus Expression Vector ◽

Golden Gate Cloning

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Distribution of Restriction Enzyme Recognition Sequences on Broad Host Range Plasmid RP4: Molecular and Evolutionary Implications

Journal of Molecular Biology ◽

10.1006/jmbi.1996.0261 ◽

1996 ◽

Vol 258 (3) ◽

pp. 447-456 ◽

Cited By ~ 25

Author(s):

Brian M. Wilkins ◽

Paul M. Chilley ◽

Angela T. Thomas ◽

Michael J. Pocklington

Keyword(s):

Host Range ◽

Restriction Enzyme ◽

Broad Host Range ◽

Broad Host Range Plasmid ◽

Enzyme Recognition

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Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02926-12 ◽

2012 ◽

Vol 79 (2) ◽

pp. 718-721 ◽

Cited By ~ 20

Author(s):

F. Heath Damron ◽

Elizabeth S. McKenney ◽

Herbert P. Schweizer ◽

Joanna B. Goldberg

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Host Range ◽

Single Copy ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Delivery Vector ◽

Inducible Gene

ABSTRACTWe describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the PBADpromoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, usingPseudomonas aeruginosaas the model host.

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A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria

Journal of Microbiological Methods ◽

10.1016/j.mimet.2017.11.024 ◽

2018 ◽

Vol 144 ◽

pp. 173-176 ◽

Cited By ~ 2

Author(s):

Rosanna C. Hennessy ◽

Line Christiansen ◽

Stefan Olsson ◽

Peter Stougaard

Keyword(s):

Gene Expression ◽

Host Range ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Dual Fluorescence ◽

Reporter System ◽

Gram Negative

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Copy-number of broad host-range plasmid R1162 is regulated by a small RNA

Nucleic Acids Research ◽

10.1093/nar/14.20.8027 ◽

1986 ◽

Vol 14 (20) ◽

pp. 8027-8046 ◽

Cited By ~ 17

Author(s):

Kyunghoon Kim ◽

Richard J. Meyer

Keyword(s):

Host Range ◽

Small Rna ◽

Copy Number ◽

Broad Host Range ◽

Broad Host Range Plasmid

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Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

Applied and Environmental Microbiology ◽

10.1128/aem.66.2.535-542.2000 ◽

2000 ◽

Vol 66 (2) ◽

pp. 535-542 ◽

Cited By ~ 10

Author(s):

Aldwin J. M. Vriesema ◽

René Brinkman ◽

Jan Kok ◽

Jacob Dankert ◽

Sebastian A. J. Zaat

Keyword(s):

Gene Expression ◽

Host Range ◽

Pathogenic Bacteria ◽

Genomic Library ◽

Regulation Of Gene Expression ◽

Broad Host Range ◽

Oral Flora ◽

Specific Stimulus ◽

Bacterial Gene ◽

Viridans Group Streptococci

ABSTRACT Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of thehydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

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