Journal: Microbial Cell Factories

Article Title: CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

doi: 10.1186/s12934-016-0595-3

Figure Lengend Snippet: Establishment of CRISPRi in PCC 7942. a Map of pSdCas9-CY’ harboring Cm R , dCas9 under P smt and eyfp under P conII , which was flanked by homology arms targeting the NSI site. T rrnb , transcriptional terminator. P smt consists of the smtA promoter and the smtB repressor. b Map of psgRNA harboring the cassette expressing Km R and sgRNA under the P J23119 promoter, which was flanked by NSII-targeting homology arms. The sgRNAs were designed to target no sequence on the PCC 7942 chromosome (Φ) or the eyfp cassette at the non-template strand of promoter (P1) or coding regions near the transcription start site (NT1 and NT2). The numbers indicate the position relative to the transcription start site. c Confocal micrographs of cells. d Flow cytometry analysis data. pSdCas9-CY’ was first transformed into PCC 7942 for cassette integration into NSI site, re-streaked, then individual psgRNA was transformed into the recombinant cells for integration into NSII site. The transformants colonies were transferred to shake flasks and cultured to OD 730 = 1–1.5 in BG-11 medium containing appropriate antibiotics

Article Snippet: Unless otherwise noted, for suspension culture S. elongatus PCC 7942 (Invitrogen) was cultivated in a 250 ml shake flask containing 40 ml BG-11 medium [ , ] with or without antibiotics (gyratory shaking at 100 rpm, with sterile air containing 0.04% CO 2 ) in a 30 °C incubator (600SR, Hipoint) with illumination from continuous cool white fluorescent light (intensity ≈ 70 μmol//m 2 s).

Techniques: Expressing, Sequencing, Flow Cytometry, Transformation Assay, Recombinant, Cell Culture

Journal: Microbial Cell Factories

Article Title: CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

doi: 10.1186/s12934-016-0595-3

Figure Lengend Snippet: Effect of CRISPRi on PCC 7942 growth and persistent transgene repression. a Mean fluorescence intensity (FI) profiles. b Growth curves. The recombinant cells were cultured in 40 ml BG-11 medium containing Km/Cm. Wild-type (WT) cells were cultured in 40 ml BG-11 without antibiotics. The cells were cultured for 21 days, during which cells were sampled for OD 730 or flow cytometry analysis

Article Snippet: Unless otherwise noted, for suspension culture S. elongatus PCC 7942 (Invitrogen) was cultivated in a 250 ml shake flask containing 40 ml BG-11 medium [ , ] with or without antibiotics (gyratory shaking at 100 rpm, with sterile air containing 0.04% CO 2 ) in a 30 °C incubator (600SR, Hipoint) with illumination from continuous cool white fluorescent light (intensity ≈ 70 μmol//m 2 s).

Techniques: Fluorescence, Recombinant, Cell Culture, Flow Cytometry

Journal: Microbial Cell Factories

Article Title: CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

doi: 10.1186/s12934-016-0595-3

Figure Lengend Snippet: Effective suppression of endogenous genes by CRISPRi. a glgc expression. b sdhA expression. c sdhB expression. PCC 7942 cells were transformed with pSdCas9 first for integration into NSI site, and were transformed again with individual psgRNA plasmids for integration into NSII site. The Km R /Cm R colonies were transferred to 40 ml BG-11 medium containing Km/Cm and cultured to OD 730 = 0.7–1.3, and 5 ml cells were sampled for qRT-PCR analysis. The expression levels were normalized to those in the WT cells

Article Snippet: Unless otherwise noted, for suspension culture S. elongatus PCC 7942 (Invitrogen) was cultivated in a 250 ml shake flask containing 40 ml BG-11 medium [ , ] with or without antibiotics (gyratory shaking at 100 rpm, with sterile air containing 0.04% CO 2 ) in a 30 °C incubator (600SR, Hipoint) with illumination from continuous cool white fluorescent light (intensity ≈ 70 μmol//m 2 s).

Techniques: Expressing, Transformation Assay, Cell Culture, Quantitative RT-PCR

Journal: Microbial Cell Factories

Article Title: CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

doi: 10.1186/s12934-016-0595-3

Figure Lengend Snippet: Effects of CRISPRi-mediated suppression on glycogen accumulation and succinate production. a Glycogen titer (µg/OD 730 ). b Succinate titer (mg/l). For glycogen analysis, Km R /Cm R cells were cultured to OD 730 ≈ 0.7–1.3, centrifuged and cultured in shake flasks containing 40 ml nitrogen-deplete (0×N) BG-11 for 2 days and subjected to glycogen analysis. To analyze succinate titer, PCC 7942 cells cultured in shake flasks to stationary phase (OD 73 ≈ 2.0) were centrifuged, and resuspended in 20 ml nitrate-free BG-11 medium. After centrifugation, the cells were resuspended in 40 ml nitrate-deplete (0×N) BG-11 medium containing Km/Cm and cultured in shake flasks. The WT cells were cultured in the nitrate-deplete (0×N) BG-11 or BG-11 (nitrate-replete) (1×N) medium

Article Snippet: Unless otherwise noted, for suspension culture S. elongatus PCC 7942 (Invitrogen) was cultivated in a 250 ml shake flask containing 40 ml BG-11 medium [ , ] with or without antibiotics (gyratory shaking at 100 rpm, with sterile air containing 0.04% CO 2 ) in a 30 °C incubator (600SR, Hipoint) with illumination from continuous cool white fluorescent light (intensity ≈ 70 μmol//m 2 s).

Techniques: Cell Culture, Centrifugation