|Title||Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria|
|Publication Type||Journal Article|
|Year of Publication||2014|
|Authors||Taton A., Unglaub F., Wright N.E, Zeng W.Y, Paz-Yepes J., Brahamsha B, Palenik B, Peterson T.C, Haerizadeh F., Golden S.S, Golden J.W|
|Journal||Nucleic Acids Research|
|Type of Article||Article|
|Keywords||anacystis-nidulans r2; binding; conjugative transfer; construction; gene-expression; plasmid; protein expression; sp; strain pcc 7120; Synechococcus; synechocystis sp|
Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bio-products, 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.
Here, we present an integrated and versatile platform for the construction of modular plasmids that can function in a broad range of cyanobacterial hosts. The modular plasmids include both autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. Overall, the goals of this study were to (i) provide a new and efficient strategy (including an in silico design portal) for the construction of broad-host-range vector systems for cyanobacteria; (ii) develop standardized devices including new or improved parts essential for the construction of cyanobacterial host-vector systems; and (iii) characterize a number of these devices in diverse cyanobacterial strains.