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Photosynthesis Research

, Volume 107, Issue 2, pp 215–221 | Cite as

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp. PCC 7002

  • Jacob H. Jacobsen
  • Lisa Rosgaard
  • Yumiko Sakuragi
  • Niels-Ulrik Frigaard
Technical Communication

Abstract

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.

Keywords

Gene inactivation Glycogen metabolism Glycogen phosphorylase Homologous recombination Natural transformation Uracil-specific excision reagent 

Abbreviations

Ap

Ampicillin

Cm

Chloramphenicol

Em

Erythromycin

Gm

Gentamicin

Km

Kanamycin

Sp

Spectinomycin

Sm

Streptomycin

USER

Uracil-specific excision reagent

Notes

Acknowledgments

This work was supported by a grant from the Danish National Advanced Technology Foundation (HTF) to N.-U. F. and a grant from the Danish Council for Independent Research | Natural Sciences (FNU) and Fuel-4-Life Consortium (University of Copenhagen) to Y. S.

Supplementary material

11120_2010_9613_MOESM1_ESM.pdf (2.5 mb)
Supplementary material 1 (PDF 2534 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Jacob H. Jacobsen
    • 1
  • Lisa Rosgaard
    • 2
  • Yumiko Sakuragi
    • 2
  • Niels-Ulrik Frigaard
    • 1
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and BiotechnologyUniversity of CopenhagenFrederiksbergDenmark

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