CyanoGate: A modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax: A Golden Gate-based toolkit for cyanobacteria

Ravendran Vasudevan, Grant A.R. Gale, Alejandra A. Schiavon, Anton Puzorjov, John Malin, Michael D. Gillespie, Konstantinos Vavitsas, Valentin Zulkower, Baojun Wang, Christopher J. Howe, David Lea-Smith, Alistair J. McCormick

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Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardised Modular Cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making i) marked/unmarked knockouts or integrations using an integrative acceptor vector, and ii) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardised MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.
Original languageEnglish
Pages (from-to)39-55
Number of pages42
JournalPlant Physiology
Early online date28 Feb 2019
Publication statusPublished - May 2019

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