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Nucleic Acids Res. 2014;42(17):e136. doi: 10.1093/nar/gku673. Epub 2014 Jul 29.

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria.

Author information

1
Division of Biological Sciences, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA aataton@gmail.com.
2
Division of Biological Sciences, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA.
3
Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA Institut de Biologie de I'Ecole Normale Supérieure, CNRS, UMR 8197, 46 rue d'Ulm, 75230 Paris, France.
4
Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093, USA.
5
Synthetic Biology Division, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008, USA.

Abstract

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

PMID:
25074377
PMCID:
PMC4176158
DOI:
10.1093/nar/gku673
[Indexed for MEDLINE]
Free PMC Article

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