Abstract
BackgroundThe cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.ResultsIn this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.ConclusionsBy reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.
Highlights
Design of the single-step double-recombination vectors A vector designed for a single-step double-recombination approach must include several essential elements: 1. an origin of replication for E. coli that is nonfunctional in Synechocystis; 2. flanking sequences homologous to Synechocystis chromosomal sites to allow stable integration of the constructs into the cyanobacterial genome; 3. the nptI-sacB double selection cassette that allows both positive and negative selection of the Synechocystis recombinants; 4. the exogenous gene of interest (GOI), split into two, partially overlapping segments (5’ GOI and 3’ GOI) that are separated by the nptI-sacB cassette; 5. a Synechocystis promoter for expression of the introduced recombinant gene(s)
When Synechocystis cells are transformed with such a vector, the initial recombination event takes place (Figure 1, upper panel) via a double crossover event between the two flanks of the construct, HR1 and HR2, and the corresponding homologous sequences present in the cyanobacterial genome
The positive selective pressure is released and the mutants are grown in the absence of selection in order to allow the second recombination event to occur (Figure 1, lower panel), without the need for a further transformation step
Summary
Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. Cyanobacteria, known as blue-green algae, are Gram-negative, photosynthetic prokaryotes They are the evolutionary ancestors of plastids [1] and the only prokaryotes that exhibit a classical circadian clock mechanism [2]. Thanks to their ability to adapt to extreme environmental conditions, cyanobacteria are widely distributed. PCC 6803 (hereafter designated as Synechocystis) is an excellent model organism, because it has a small (3.6 Mb), sequenced genome [5], is capable of spontaneous uptake of exogenous DNA and can integrate it via homologous recombination into its genome. A spontaneous glucose-tolerant mutant is available [6] that can
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