Name: spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis
Uploaded: 2018-09-11T13:30:00
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Protokollen blev vedtaget fra en tidligere publikation11. T. Z., D. Ch. og J. C. blev støttet af Ministeriet for uddannelse, Ungdom og sport i Tjekkiet inden for den nationale bæredygtighed Program jeg (NPU jeg), give nummer LO1415. J. C. blev også støttet af GA CR, tilskud antallet 18-24397S. Adgang til instrumenter og andre faciliteter blev støttet af den tjekkiske forskningsinfrastruktur for Systembiologi C4SYS (projekt ingen LM2015055). M. A. S. blev støttet af en bevilling fra den russiske Science Foundation [nr. 14-14-00904].

1. cyanobakterier dyrkning

<ol><li>Dyrke Synechocystis celler i Erlenmeyerkolben kolber eller i photobioreactors10,19 i "buffered" BG11 medium20 at fastholde en pH på < 10 (f.eks.ved hjælp af 17 mM HEPES10). Bemærk: Standard dyrkning betingelser kræver en kontrolleret temperatur (typisk 30 ° C, den optimale temperatur er 35 ° C)21, belysning (typisk et hvi...

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Sequence determination of the entire genome and assignment of potential protein-coding regions.</a> DNA Research. 3, 109-136 (1996).</li><li>Grigorieva, G., Shestakov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+in+the+cyanobacterium+Synechocystis+sp+6803.">Transformation in the cyanobacterium Synechocystis sp 6803.</a> FEMS Microbiology Letters. 13 (4), 367-370 (1982).</li><li>Zav&#345;el, T., Sinetova, M. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycobilisomes+from+Cyanobacteria.">Phycobilisomes from Cyanobacteria.</a> Handbook on Cyanobacteria: Biochemistry, Biotechnology and Applications. , 105-160 (2009).</li><li>Six, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+evolution+of+phycobilisomes+in+marine+Synechococcus+spp.:+A+comparative+genomics+study.">Diversity and evolution of phycobilisomes in marine Synechococcus spp.: A comparative genomics study.</a> Genome Biology. 8 (12), (2007).</li><li>Sinetova, M. A., &#268;erven&#253;, J., Zav&#345;el, T., Nedbal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+dynamics+and+constraints+of+batch+culture+growth+of+the+cyanobacterium+Cyanothece+sp.+ATCC+51142.">On the dynamics and constraints of batch culture growth of the cyanobacterium Cyanothece sp. ATCC 51142.</a> Journal of Biotechnology. 162 (1), (2012).</li><li>Stanier, R. Y., Kunisawa, R., Mandel, M., Cohen-Bazire, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+unicellular+blue-green+algae+(order+Chroococcales).">Purification and properties of unicellular blue-green algae (order Chroococcales).</a> Bacteriological Reviews. 35 (2), 171-205 (1971).</li><li>Zav&#345;el, T., Sinetova, M. 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PCC 6803 autotrophic growth in a flat-panel photobioreactor.</a> Engineering in Life Sciences. 15 (1), 122-132 (2015).</li><li>Hemlata, G., Fareha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Anabaena+sp.+nccu-9+with+special+reference+to+phycocyanin.">Studies on Anabaena sp. nccu-9 with special reference to phycocyanin.</a> Journal of Algal Biomass Utilization. 2 (1), 30-51 (2011).</li><li>Rito-Palomares, M., Nuez, L., Amador, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+application+of+aqueous+two-phase+systems+for+the+development+of+a+prototype+process+for+c-phycocyanin+recovery+from+Spirulina+maxima.">Practical application of aqueous two-phase systems for the development of a prototype process for c-phycocyanin recovery from Spirulina maxima.</a> Journal of Chemical Technology &amp; Biotechnology. 76 (12), 1273-1280 (2001).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenium-Containing+Allophycocyanin+Purified+from+Selenium-Enriched+Spirulina+platensis+Attenuates+AAPH-Induced+Oxidative+Stress+in+Human+Erythrocytes+through+Inhibition+of+ROS+Generation.">Selenium-Containing Allophycocyanin Purified from Selenium-Enriched Spirulina platensis Attenuates AAPH-Induced Oxidative Stress in Human Erythrocytes through Inhibition of ROS Generation.</a> Journal of Agricultural and Food Chemistry. 59 (16), 8683-8690 (2011).</li><li>Nedbal, L., Trt&#237;lek, M., Cerven&#253;, J., Kom&#225;rek, O., Pakrasi, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photobioreactor+system+for+precision+cultivation+of+photoautotrophic+microorganisms+and+for+high-content+analysis+of+suspension+dynamics.">A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics.</a> Biotechnology and Bioengineering. 100 (5), 902-910 (2008).</li><li>Zav&#345;el, T., Knoop, H., Steuer, R., Jones, P. R., &#268;erven&#253;, J., Trt&#237;lek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+evaluation+of+ethylene+production+in+the+recombinant+cyanobacterium+Synechocystis+sp.+PCC+6803+harboring+the+ethylene-forming+enzyme+by+membrane+inlet+mass+spectrometry.">A quantitative evaluation of ethylene production in the recombinant cyanobacterium Synechocystis sp. PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry.</a> Bioresource Technology. 202, 142-151 (2016).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Lawrenz, E., Fedewa, E. J., Richardson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+protocols+for+the+quantification+of+phycobilins+in+aqueous+phytoplankton+extracts.">Extraction protocols for the quantification of phycobilins in aqueous phytoplankton extracts.</a> Journal of Applied Phycology. 23 (5), 865-871 (2011).</li><li>Lea-Smith, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phycobilisome-Deficient+Strains+of+Synechocystis+sp.+PCC+6803+Have+Reduced+Size+and+Require+Carbon-Limiting+Conditions+to+Exhibit+Enhanced+Productivity.">Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.</a> Plant Physiology. 165 (2), 705-714 (2014).</li><li>Seo, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isolation+of+phycocyanin+from+Spirulina+platensis+associated+with+high-pressure+extraction+process.">Stable isolation of phycocyanin from Spirulina platensis associated with high-pressure extraction process.</a> International Journal of Molecular Sciences. 14 (1), 1778-1787 (2013).</li><li>Touloupakis, E., Cicchi, B., Torzillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioenergetic+assessment+of+photosynthetic+growth+of+Synechocystis+sp.+PCC+6803+in+continuous+cultures.">A bioenergetic assessment of photosynthetic growth of Synechocystis sp. PCC 6803 in continuous cultures.</a> Biotechnology for Biofuels. 8 (1), 133 (2015).</li><li>Touloupakis, E., Cicchi, B., Benavides, A. M. 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Denne protokol beskriver en enkel, hurtig og reproducerbar metode til kvantificering af phycobiliprotein indhold i model cyanobacterium Synechocystis. Flere metoder til celle homogenisering, protein udvinding og phycocyanin og allophycocyanin kvantificering sammenlignes, og den endelige protokol repræsenterer en kombination af de optimale skridt af hver enkelt procedure. Som repræsentative data, var indholdet af fykobiliproteiner kvantificeret i Synechocystis celler under stigende lysintensitet. Selvo...

fPhycobiliproteins er vandopløselige pigment-protein komplekser, der udgør væsentlige bestanddele af de lys-høst antenner i prokaryote cyanobakterier (Cyanophyta) og flere eukaryote taxa (Glaucophyta, Rhodophyta , og Cryptophyta)1. De opstår hovedsagelig som Supramolekylær komplekser kaldes phycobilisomes og de er typisk knyttet til overfladen af de fotosyntetiske membraner i stromale side, med undtagelse af Cryptophyta, hvor fykobiliproteiner er lokaliseret i den tylakoid lumen2. Fire typer af fykobiliproteiner er blevet identificeret op til dato: core allophycocyanin og den perifere phycocyanin, phycoerythrin og phycoerythrocyanin1. Som de vigtigste lys-høst komplekser repræsenterer phycobilisomes en af de afgørende faktorer for alger og cyanobakterier masse kulturer produktivitet. Det er blevet påvist, at phycobilisomes afkortning kan forbedre biomasse akkumulering under stærkt lys3. På den anden side under beskedne eller lav irradians resulterede antenne trunkering i vækst og biomasse ophobning reduktion3,4. Fykobiliproteiner bruges kommercielt som mad farvestoffer, lægemidler og tilsætningsstoffer i den kosmetiske industri, samt fluorescens sonder med programmer i flowcytometri, fluorescerende immunassays og Fluorescens mikroskopi5.
Denne protokol fokuserer på den kvantitative bestemmelse af fykobiliproteiner i model cyanobacterium Synechocystis. Cyanobakterier er de tidligste iltdannende fotosyntetiske autotrofer; de har danner Jordens biosfære for mere end 2,4 milliarder år6. De spiller en afgørende rolle i globale biogeokemiske kredsløb af kvælstof, kulstof, ilt og andre elementer. Blandt cyanobakterier, en encellet stamme Synechocystis fik en unik position, da det var den første cyanobacterium med det hele genom sekventeret7,8, det er naturligvis udklappelige af eksogene DNA9, og det udfører stabilt og relativt hurtig vækst10,11. I Synechocystis, antenne kernekomponenter, allophycocyanin, er forbundet med integreret Membranproteiner, og de vedlagte phycocyanin ligger på tylakoid membran periferien.
Flere metoder til phycobiliprotein udvinding og kvantificering er sammenlignet i denne protokol. Den endelige ekstraktionsmetode blev anvendt til Synechocystissamt til andre stammer, cyanobakterier, herunder Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira SP., og stenflader sp., og det var også med held anvendes til rødalger Porphyridium cruentum. Metoden udviklet i denne protokol kan derfor betragtes som en universel metode til phycobiliprotein udvinding. Selv om nogle af de testede udvindingsmetoder resulterede i højere total protein udbytter, her beskrevne ekstraktionsmetode forudsat det højeste phycobiliprotein giver sammen med det laveste indhold af klorofyl en rest i den phycobiliprotein ekstrakt. At reducere indholdet af klorofyl en var afgørende for den korrekte phycocyanin og allophycocyanin spektrofotometriske kvantificering.
Absorptionsspektra phycobiliprotein kan variere betydeligt mellem forskellige alger og cyanobakterier arter12,13,14,15,16,17 og endda blandt flere stammer af en enkelt cyanobakterier slægten18. Derfor er bestemte bølgelængder og absorption koefficienter som anvendes til bestemmelse af phycocyanin og allophycocyanin i Synechocystis ikke generelt gælder for andre stammer. Derudover indeholder Synechocystis ikke phycoerythrin og phycoerythrocyanin, der findes i nogle andre alger og cyanobakterier. Med henblik på bestemmelse af fykobiliproteiner i stammer end Synechocystisanbefales det at evaluere de spektrofotometriske ligninger for hver stamme individuelt.
Selv om protokollen indeholder to længere skridt (natten frysetørring af cellulære pellets og 1 times protein udvinding), er den samlede arbejdsmængde tid til fykobiliproteiner kvantificering ikke længere end 2 timer.

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			<td height="21" style="height:21px;width:407px;">Synechocystis sp. PCC 6803</td>
			<td style="width:417px;">Institut Pasteur, Paris, France</td>
			<td style="width:137px;">6803</td>
			<td style="width:627px;">Cyanobacterium strain</td>
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			<td height="21" style="height:21px;">Roti-CELL PBS</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe, Germany</td>
			<td>9143.1</td>
			<td>Phosphate-Buffered Saline (PBS) solution, pH 7.4</td>
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			<td height="21" style="height:21px;">Eppendorf safe-lock tubes&#160;</td>
			<td style="width:417px;">Eppendorf, Hamburk, Germany</td>
			<td>30120086</td>
			<td>Safe-lock tubes 1.5 ml</td>
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			<td height="21" style="height:21px;">VWR 80-Place Storage System</td>
			<td style="width:417px;">VWR International, Radnor, Pennsylvania, USA</td>
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			<td height="21" style="height:21px;">Rotina 420R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td>4701</td>
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		<tr height="21">
			<td height="21" style="height:21px;">LCexv 4010</td>
			<td>Liebherr, Bulle, Switzerland</td>
			<td>9005382197172</td>
			<td>Refrigerator and freezer -20&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Revco ExF -86&#176;C Upright Ultra-Low Temperature Freezer</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">EXF24086V&#160;</td>
			<td>Freezer -80&#160;&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolSafe</td>
			<td>LaboGene, Liller&#248;d, Denmark</td>
			<td>7.001.000.615</td>
			<td>Freeze dryer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-2600</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>UV-2600</td>
			<td>Spectrophotometer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hellma absorption cuvettes, semi Micro</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z600288&#160;</td>
			<td>VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silamat S6</td>
			<td>Ivoclar Vivadent, Schaan, Liechtenstein</td>
			<td>602286WU</td>
			<td>Homogenizer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solid-glass beads</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>Z273627</td>
			<td>Glass bead of the diameter 2&#160;mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPA225D-0CE</td>
			<td>Sartorius AG, G&#246;ttingen, Germany</td>
			<td>SECURA225D-1OBR</td>
			<td>Analytical balances</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C-Phycocyanin from Spirulina sp.&#160;</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P2172</td>
			<td>Phycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allophycocyanin</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>A7472</td>
			<td>Allophycocyanin standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Bicinchoninic Acid Kit&#160;</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">BCA1, B9643</td>
			<td>Complete kit for total proteins determination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">AlgaeTron&#160;</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">AG 130-ECO&#160;</td>
			<td>Cultivation chamber for E. flasks, with controllable light and atmosphere</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Photobioreactor</td>
			<td style="width:417px;">Photon System Instruments Ltd., Dr&#225;sov, Czech Republic</td>
			<td style="width:137px;">FMT-150</td>
			<td>Cultivation equipment for cyanobacteria and algae with completely controllable environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer&#160;</td>
			<td style="width:417px;">Nexcelom Bioscience, Lawrence, Massachusetts, USA</td>
			<td style="width:137px;">Auto M10</td>
			<td>Cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Corning 15 mL centrifuge tubes</td>
			<td style="width:417px;">Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td style="width:137px;">CLS430791&#160;</td>
			<td>15 ml Centrifuge tube for dry weigth sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">Herasafe KS</td>
			<td style="width:417px;">Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td style="width:137px;">51024579</td>
			<td>Laminar flow hood</td>
		</tr>
	</tbody>
</table>

spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis

Dette er en simpel protokol til kvantitativ bestemmelse af phycobiliprotein indhold i model cyanobacterium Synechocystis. Fykobiliproteiner er de vigtigste komponenter i phycobilisomes, de store lys-høst antenner i cyanobakterier og flere alger taxa. Phycobilisomes af Synechocystis indeholder to fykobiliproteiner: phycocyanin og allophycocyanin. Denne protokol beskriver en enkel, effektiv og pålidelig metode til kvantitativ bestemmelse af både phycocyanin og allophycocyanin i denne model cyanobacterium. Vi sammenlignede flere metoder til phycobiliprotein ekstraktion og spektrofotometrisk kvantificering. Ekstraktionsmetode som beskrevet i denne protokol blev også anvendt til andre cyanobakterier stammer som Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., og stenflader sp., samt om rødalger Porphyridium cruentum. Men udryddelse koefficienter af specifikke fykobiliproteiner fra forskellige taxa kan variere og det anbefales derfor, at validere metoden spektrofotometriske kvantificering for hver enkelt stamme individuelt. Protokollen kræver lidt tid og kan udføres i alle standard life science laboratorier, da det kræver kun standard udstyr.

For de oprindelige metode tests, Synechocystis blev dyrket som batch kulturer i Erlenmeyerkolben kolber på en shaker i BG11 dyrkning medium20 (suppleret med 17 mM HEPES) på 25 ° C, under et varmt hvidt lys med en intensitet på 50 µmol (fotoner) / (m 2·s) og med 1% CO2 i de dyrkningsbaserede atmosfære. Under dyrkning, kulturer var udtaget til sikker-lock rør og centrifugeres (15.000 x g laboratorium temperatur i 5 min)...

Watch this Scientific Journal Video about Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis at JoVE.com

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Vi præsenterer her, en protokol til at bestemme kvantitativt phycobiliprotein indhold i cyanobacterium Synechocystis ved hjælp af en Spektrofotometrisk metode. Ekstraktionsmetode blev også anvendt til andre cyanobakterier og alger stammer; på grund af variationer i pigment absorptionsspektre, er det imidlertid nødvendigt at teste de spektrofotometriske ligninger for hver stamme individuelt.

Spektrofotometrisk bestemmelse af Phycobiliprotein indhold i Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

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JoVE Journal

Biochemistry

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

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