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

被引:14
作者
Jacobsen, Jacob H. [1 ]
Rosgaard, Lisa [2 ]
Sakuragi, Yumiko [2 ]
Frigaard, Niels-Ulrik [1 ]
机构
[1] Univ Copenhagen, Dept Biol, DK-2200 Copenhagen, Denmark
[2] Univ Copenhagen, Dept Plant Biol & Biotechnol, Plant Mol Biol Lab, VKR Res Ctr Proact Plants, DK-1871 Frederiksberg, Denmark
关键词
Gene inactivation; Glycogen metabolism; Glycogen phosphorylase; Homologous recombination; Natural transformation; Uracil-specific excision reagent; SYNECHOCYSTIS SP PCC-6803; NATURAL TRANSFORMATION; CLONING; MUTAGENESIS; FUSION;
D O I
10.1007/s11120-010-9613-1
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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.
引用
收藏
页码:215 / 221
页数:7
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