title: A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.
authors.forename: Stefania
authors.initials: S
authors.lastname: Viola
authors.forename: Thilo
authors.initials: T
authors.lastname: Rühle
authors.affiliation: Department Biology I, Ludwig-Maximilians-Universität München, Großhaderner Str, 2, Planegg, Martinsried D-82152, Germany. leister@lmu.de.
authors.forename: Dario
authors.initials: D
authors.lastname: Leister
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D059765:Homologous Recombination; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins