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

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.

Anchoring a Plant Cytochrome P450 via PsaM to the Thylakoids in Synechococcus sp. PCC 7002: Evidence for Light-Driven Biosynthesis

“…The 831 bp flanking region upstream and 791 bp flanking region downstream of the psaM gene were amplified from the Synechococcus sp. PCC 7002 gDNA using primer pair b and c, respectively, and the npt cassette, conferring kanamycin resistance, was amplified from the pJHJ08 vector ([25], gift from associate professor Yumiko Sakuragi) using primer pair a by PCR (primers are listed in Table 1). The fragments were cloned into the pUC57 construct in three consecutive cloning steps at EcoRI, BamHI and BspEI sites as illustrated in Figure S1 to yield the psaM-CYP79A1 fusion construct.…”

“…Using PCR, a kanamycin resistance cassette was amplified from a vector containing the cassette ( npt cassette from pJHJ08, [25]) and flanking regions of the psaM gene were amplified from Synechococcus sp. PCC 7002 gDNA.…”

An Innate Immunity Pathway in the Moss Physcomitrella patens

“…The respective genes were cloned into the resulting USER cloning vector pMBLU by PCR amplification of their 59 upstream and 39 downstream regions with USER compatible primers. These two PCR products were combined in a single, four-fragment USER cloning step into pMBLU cut with PacI and AsiSI and nicked with Nt.BbvCI (Jacobsen et al, 2011). GFP and a nos terminator were cloned in the pMBLU KpnI site to make the USER cloning vector pMBLU-GFP for tagging proteins with a C-terminal GFP.…”

Computational evaluation of Synechococcus sp. PCC 7002 metabolism for chemical production

“…Compared to other cyanobacteria strains, it has a fast doubling time (∼3.5 h compared to ∼12–24 h) indicating a high inherent metabolic rate. Synechococcus 7002 also can grow under high light conditions (up to ∼4.5 mE/m 2 /s compared to its optimal light intensity of 250 μE/m 2 /s) [10], can grow in salt water (obviating a requirement for fresh water) [11] and can be genetically manipulated [12–14]. In addition, the strain's genome has been sequenced and a variety of high‐throughput experimental and computational tools have been used to evaluate it, including gene expression and genome‐scale metabolic modeling [15–17].…”