Protocol

Photosynthesis Research Protocols

Volume 684 of the series Methods in Molecular Biology pp 273-293

Date:

Expression of Genes in Cyanobacteria: Adaptation of Endogenous Plasmids as Platforms for High-Level Gene Expression in Synechococcus sp. PCC 7002

  • Yu XuAffiliated withDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University
  • , Richard M. AlveyAffiliated withDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University
  • , Patrick O. ByrneAffiliated withDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University
  • , Joel E. GrahamAffiliated withDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University
  • , Gaozhong ShenAffiliated withDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University
  • , Donald A. BryantAffiliated withDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University

* Final gross prices may vary according to local VAT.

Get Access

Abstract

Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.

Key words

Synechococcus Gene expression Affinity-tag Homologous recombination Cyanobacteria Transformation Plasmid