蓝藻集胞藻PCC 6803中糖原和胞外葡萄糖估测

Md. Rezaul Islam Khan; Yushu Wang; Shajia Afrin; Lin He; Gang Ma

doi:10.21769/BioProtoc.2826

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Peer-reviewed

Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803

蓝藻集胞藻PCC 6803中糖原和胞外葡萄糖估测

MK Md. Rezaul Islam Khan

YW Yushu Wang

SA Shajia Afrin

LH Lin He

GM Gang Ma email

发布: 2018年05月05日第8卷第9期 DOI: 10.21769/BioProtoc.2826 浏览次数: 6465

评审: Anonymous reviewer(s)

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Abstract

Cyanobacteria, which have the extraordinary ability to grow using sunlight and carbon dioxide, are emerging as a green host to produce value-added products. Exploitation of this highly promising host to make products may depend on the ability to modulate the glucose metabolic pathway; it is the key metabolic pathway that generates intermediates that feed many industrially important pathways. Thus, before cyanobacteria can be considered as a leading source to produce value-added products, we must understand the interaction between glucose metabolism and other important cellular activities such as photosynthesis and chlorophyll metabolism. Here we describe reproducible and reliable methods for measuring extracellular glucose and glycogen levels from cyanobacteria.

Keywords: Extracellular glucose (胞外葡萄糖)

Glycogen (糖原)

Cyanobacteria (蓝藻)

Synechocystis sp. PCC 6803 (集胞藻PCC 6803)

Background

Cyanobacteria have a light-dark cycle in their natural habitat. In the light, their metabolism is centered on photosynthesis, the Calvin cycle, glycolysis and the TCA cycle with N-assimilation; carbon is stored as glycogen. In the dark, glycogen is metabolized through glycolysis and the oxidative pentose phosphate (OPP) pathway, the oxidative and reductive branches of the TCA cycle, and the C4 cycle (Nagarajan et al., 2014). Thus, the shift from dark to light or light to dark drives metabolic reprogramming.

In the laboratory, the addition of glucose to the culture media also impacts cyanobacteria metabolic programs. For example, nutritional and environmental conditions influence how the cyanobacterium Synechocystis metabolizes glucose; Synechocystis metabolizes glucose differently in photoautotrophic, heterotrophic and mixotrophic conditions. Previous studies reported that some strains of Synechocystis are light-dependent and glucose tolerant (Anderson and McIntosh, 1991). Light-activated heterotrophic growth (LAHG) conditions are characterized by the presence of glucose and growth in the dark with a pulse of white or blue light for at least 5-15 min per day. However, some strains of Synechocystis are glucose intolerant, meaning that they cannot grow in the presence of glucose in the dark. In summary, the addition of glucose to the culture media of Synechocystis has been reported to bring physiological and metabolic changes such as pigmentation (Ryu et al., 2004), carbon metabolism (Lee et al., 2007; Takahashi et al., 2008), phosphorylation patterns (Bloye et al., 1992), carbon dioxide uptake (Kaplan and Reinhold, 1999), and oxidative stress generation (Narainsamy et al., 2013).

To identify the utility of cyanobacteria to produce natural product, growing cyanobacteria in large-scale is a prerequisite. For growing cyanobacteria efficiently, it’s important to characterize the direct impact of common environmental factors such as light and temperature on glucose metabolism. Here, we present an accurate, reproducible, and reliable method to quantify extracellular glucose and glycogen levels of cyanobacteria, we belelive that this method will help determine the utility of cyanobacteria as a source for engineering natural products.

Materials and Reagents

Pipette tips (20 µl-1 ml, autoclaved)
Aluminum foil
1.5 and 2 ml Eppendorf tubes (autoclaved)
0.45 µm filter
Cyanobacteria Synechocystis sp. PCC 6803 (WT, mutant D95 & C95)
Note: For more information about these strains, please see Data analysis A4.
Sulfuric acid (ACS reagent, Sigma-Aldrich, catalog number: S1526 )
Nitrogen
Ethanol (Sigma-Aldrich, catalog number: 362808-1L )
Glycogen (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: R0561 )
Amyloglucosidase (Sigma-Aldrich, catalog number: 10115-1G-F )
Sodium carbonate (Na₂CO₃) (Sangon Biotech, catalog number: ST0840 )
Sodium nitrate (NaNO₃) (Sinopharm Chemical Reagent, catalog number: 10019918 )
Hydrochloric acid (HCl) (Sinopharm Chemical Reagent, catalog number: 10011018 )
Sodium hydroxide (NaOH) (Sangon Biotech, catalog number: SB0617 )
Potassium phosphate dibasic (K₂HPO₄) (Sangon Biotech, catalog number: PB0447 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sangon Biotech, catalog number: MT0864 )
Ferric ammonium sulphate (Sangon Biotech, catalog number: A502657 )
Citric acid (Sangon Biotech, catalog number: C0529 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sangon Biotech, catalog number: CT1331 )
EDTA-Na₂ (Sangon Biotech, catalog number: E0105 )
Boric acid (H₃BO₃) (Sangon Biotech, catalog number: BB0044 )
Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Sangon Biotech, catalog number: A500331 )
Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sangon Biotech, catalog number: A602906 )
Sodium molybdate dehydrate (Na₂MoO₄·2H₂O) (Sangon Biotech, catalog number: SB0865 )
Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Sangon Biotech, catalog number: A501425 )
Cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O) (Sangon Biotech, catalog number: CB7774 )
Sodium thiosulfate anhydrous (Na₂S₂O₃) (Sangon Biotech, catalog number: S1712 )
D-glucose (Sangon Biotech, catalog number: 501991 )
Glucose standard solution (Sigma-Aldrich, catalog number: G3285 )
Benzoic acid (Sinopharm Chemical Reagent, catalog number: 30018615 )
Glucose oxidase/peroxidase (Sigma-Aldrich, catalog number: G3660 )
o-Dianisidine reagent (Sigma-Aldrich, catalog number: D2679 )
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P6310 )
Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Sangon Biotech, catalog number: TB0927 )
BG-11 media (see Recipes)
D-Glucose (see Recipes)
Assay reagent (see Recipes)
30% (w/v) KOH (see Recipes)
100 mM sodium acetate (pH 4.5) (see Recipes)

Equipment

Conical flasks (100, 200, 500 ml) (SHUNIU, Chengdu, China)
Vortex (FINE PCR, model: Finevortex )
Centrifuge machine, unrefrigerated, maximum speed 17,000 x g, with rotor for microtubes (Thermo Fisher Scientific, model: Heraeues^TM Pico^TM 17 )
Glass test tubes 18 x 150 mm
Spectrophotometer (METASH, model: V-5600 )
1 ml glass cuvettes (METASH)
Pipettes (20 µl, 200 µl, 1 ml, 5 ml) (Gilson, France)
Water baths (temperature set at 37 ± 1 °C; 60 °C; 92 °C) (Meier, model: XMTD-204 )
Shaking light-incubator (light:up to 150 µmoles/m²/sec; temperature: 20-50 °C) (Shanghai Zhichu, model: ZQWY-200G )
Freeze dryer (Labconco, model: FreeZone Plus 6 )
Oven, 60 °C (Boxun, model: GZX-9140MBE )
Autoclave (Boxun, model: YXQ-LS-100SII )
pH meter (Mettler Toledo, model: FE 20 )
Swimming holders

Software

GraphPad PRISM (Version 5.01)

Procedure

English

中文翻译

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如何引用

Khan, M. R. I., Wang, Y., Afrin, S., He, L. and Ma, G. (2018). Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803. Bio-protocol 8(9): e2826. DOI: 10.21769/BioProtoc.2826.