caa a22 4500 44588987X CHVBK 20180317145612.0 cr unu---uuuuu 170323e20110201xx s 000 0 eng 10.1007/s11120-010-9613-1 doi (NATIONALLICENCE)springer-10.1007/s11120-010-9613-1 One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp. PCC 7002 [Elektronische Daten] [Jacob Jacobsen, Lisa Rosgaard, Yumiko Sakuragi, Niels-Ulrik Frigaard] 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. Springer Science+Business Media B.V., 2011 Gene inactivation nationallicence Glycogen metabolism nationallicence Glycogen phosphorylase nationallicence Homologous recombination nationallicence Natural transformation nationallicence Uracil-specific excision reagent nationallicence Ap : Ampicillin nationallicence Cm : Chloramphenicol nationallicence Em : Erythromycin nationallicence Gm : Gentamicin nationallicence Km : Kanamycin nationallicence Sp : Spectinomycin nationallicence Sm : Streptomycin nationallicence USER : Uracil-specific excision reagent nationallicence Jacobsen Jacob Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen, Denmark aut Rosgaard Lisa Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark aut Sakuragi Yumiko Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark aut Frigaard Niels-Ulrik Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen, Denmark aut Photosynthesis Research Springer Netherlands 107/2(2011-02-01), 215-221 0166-8595 107:2<215 2011 107 11120 https://doi.org/10.1007/s11120-010-9613-1 text/html Onlinezugriff via DOI 1 brief-communication jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11120-010-9613-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Jacobsen Jacob Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen, Denmark aut NATIONALLICENCE 700 1- Rosgaard Lisa Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark aut NATIONALLICENCE 700 1- Sakuragi Yumiko Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark aut NATIONALLICENCE 700 1- Frigaard Niels-Ulrik Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200, Copenhagen, Denmark aut NATIONALLICENCE 773 0- Photosynthesis Research Springer Netherlands 107/2(2011-02-01), 215-221 0166-8595 107:2<215 2011 107 11120 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer