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Biochemistry

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Published: September 11th, 2018

DOI:

10.3791/58076

1Department of Adaptive Biotechnologies, Global Change Research Institute, Czech Academy of Sciences, 2Department of Plant Physiology, Faculty of Science, Masaryk University, 3Laboratory of Intracellular Regulation, Institute of Plant Physiology, Russian Academy of Sciences

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

fPhycobiliproteins are water-soluble pigment-protein complexes that represent major components of the light-harvesting antennae in prokaryotic cyanobacteria (Cyanophyta) and several eukaryotic taxa (Glaucophyta, Rhodophyta, and Cryptophyta)1. They occur mainly as supramolecular complexes called phycobilisomes and they are typically attached to the surface of the photosynthetic membranes on the stromal side, with the exception of Cryptophyta, where the phycobiliproteins are localized in the thylakoid lumen2. Four types of phycobiliproteins have been identified up to date: the c....

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1. Cyanobacteria Cultivation

  1. Cultivate Synechocystis cells in Erlenmeyer flasks or in photobioreactors10,19 in buffered BG11 medium20 to maintain a pH of < 10 (e.g., using 17 mM HEPES10).
    NOTE: Standard cultivation conditions require a controlled temperature (typically, 30 °C, the optimal temperature is 35 °C)21, illumination (typical.......

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For the initial method tests, Synechocystis was cultivated as batch cultures in Erlenmeyer flasks on a shaker in BG11 cultivation medium20 (supplemented with 17 mM HEPES) at 25 °C, under a warm white light of an intensity of 50 µmol(photons)/(m2·s) and with 1% CO2 in the culturing atmosphere. During the cultivation, the cultures were sampled to safe-lock tubes and centrifuged (15,000 x g at laboratory temperatu.......

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This protocol describes a simple, fast, and reproducible method for the quantification of phycobiliprotein content in the model cyanobacterium Synechocystis. Several methods of cell homogenization, protein extraction, and phycocyanin and allophycocyanin quantification are compared, and the final protocol represents a combination of the optimal steps of every single procedure. As representative data, the content of phycobiliproteins was quantified in Synechocystis cells under increasing light intensity. .......

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The protocol was adopted from a previous publication11. T. Z., D. Ch., and J. Č. were supported by the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (NPU I), grant number LO1415. J. Č. was also supported by GA CR, Grant number 18-24397S. Access to instruments and other facilities was supported by the Czech research infrastructure for systems biology C4SYS (project no LM2015055). M. A. S. was supported by a grant from the Russian Science Foundation [no. 14-14-00904].

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Name Company Catalog Number Comments
Synechocystis sp. PCC 6803 Institut Pasteur, Paris, France 6803 Cyanobacterium strain
Roti-CELL PBS Carl Roth GmbH + Co. KG, Karlsruhe, Germany 9143.1 Phosphate-Buffered Saline (PBS) solution, pH 7.4
Eppendorf safe-lock tubes  Eppendorf, Hamburk, Germany 30120086 Safe-lock tubes 1.5 ml
VWR 80-Place Storage System VWR International, Radnor, Pennsylvania, USA 30128-282 Holder for safe-lock tubes 
RAININ 100 µl -1000 µl  Mettler-Toledo, Columbus, Ohio, USA 17014382 Pipette
GP-LTS-A-1000µL-/F-768/8 Mettler-Toledo, Columbus, Ohio, USA 30389272 Pipette tips
Rotina 420R Hettich, Kirchlengern, Germany 4701 Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes
LCexv 4010 Liebherr, Bulle, Switzerland 9005382197172 Refrigerator and freezer -20 °C
Revco ExF -86°C Upright Ultra-Low Temperature Freezer Thermo Fisher Scientific, Waltham, Massachusetts, USA EXF24086V  Freezer -80 °C
CoolSafe LaboGene, Lillerød, Denmark 7.001.000.615 Freeze dryer 
UV-2600 Shimadzu, Kyoto, Japan UV-2600 Spectrophotometer 
Hellma absorption cuvettes, semi Micro Sigma-Aldrich, St. Louis, Missouri, USA Z600288  VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm 
Silamat S6 Ivoclar Vivadent, Schaan, Liechtenstein 602286WU Homogenizer 
Solid-glass beads Sigma-Aldrich, St. Louis, Missouri, USA Z273627 Glass bead of the diameter 2 mm
CPA225D-0CE Sartorius AG, Göttingen, Germany SECURA225D-1OBR Analytical balances
C-Phycocyanin from Spirulina sp.  Sigma-Aldrich, St. Louis, Missouri, USA P2172 Phycocyanin standard
Allophycocyanin Sigma-Aldrich, St. Louis, Missouri, USA A7472 Allophycocyanin standard
Bicinchoninic Acid Kit  Sigma-Aldrich, St. Louis, Missouri, USA BCA1, B9643 Complete kit for total proteins determination
AlgaeTron  Photon System Instruments Ltd., Drásov, Czech Republic AG 130-ECO  Cultivation chamber for E. flasks, with controllable light and atmosphere
Photobioreactor Photon System Instruments Ltd., Drásov, Czech Republic FMT-150 Cultivation equipment for cyanobacteria and algae with completely controllable environment
Cellometer  Nexcelom Bioscience, Lawrence, Massachusetts, USA Auto M10 Cell counter
Corning 15 mL centrifuge tubes Sigma-Aldrich, St. Louis, Missouri, USA CLS430791  15 ml Centrifuge tube for dry weigth sampling
Herasafe KS Thermo Fisher Scientific, Waltham, Massachusetts, USA 51024579 Laminar flow hood

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