Name: spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis
Uploaded: 2018-09-11T13:30:00
Description: Watch this Scientific Journal Video about Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis at JoVE.com

Protokollen ble innført fra en tidligere publikasjon11. T. Z., D. Ch. og J. Č. ble støttet av departementet for utdanning, ungdom og sport Tsjekkias nasjonale bærekraft programmet jeg (NPU jeg), gi nummer LO1415. J. Č. ble også støttet av GA CR, bevilgning nummer 18-24397S. Tilgang til instrumenter og andre fasiliteter ble støttet av tsjekkiske forskning infrastrukturen for systemer biologi C4SYS (project ikke LM2015055). M. A. S. ble støttet av et stipend fra russisk Science Foundation [nr. 14-14-00904].

1. Cyanobakterie dyrking

<ol><li>Dyrke Synechocystis celler i Erlenmeyer flasker eller photobioreactors10,19 i bufret BG11 middels20 å opprettholde en pH på < 10 (f.eksbruker 17 mM HEPES10). Merk: Standard dyrking betingelser krever en kontrollert temperatur (vanligvis 30 ° C, den optimale temperaturen er 35 ° C)21, belysning (vanligvis et hvitt lys med en i...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+location+of+the+phycobiliprotein+light-harvesting+pigment+in+the+Cryptophyceae.">Unique location of the phycobiliprotein light-harvesting pigment in the Cryptophyceae.</a> Journal of Phycology. 25 (3), 412-419 (1989).</li><li>Kirst, H., Formighieri, C., Melis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+photosynthetic+efficiency+and+culture+productivity+in+cyanobacteria+upon+minimizing+the+phycobilisome+light-harvesting+antenna+size.">Maximizing photosynthetic efficiency and culture productivity in cyanobacteria upon minimizing the phycobilisome light-harvesting antenna size.</a> Biochimica et Biophysica Acta - Bioenergetics. 1837 (10), 1653-1664 (2014).</li><li>Page, L. E., Liberton, M., Pakrasi, H. 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Sequence features in the 1 Mb region from map positions 64% to 92% of the genome.</a> DNA Research. 2, 191-198 (1995).</li><li>Kaneko, T., 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=Sequence+analysis+of+the+genome+of+the+unicellular+cyanobacterium+Synechocystis+sp.+strain+PCC6803.+II.+Sequence+determination+of+the+entire+genome+and+assignment+of+potential+protein-coding+regions.">Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. 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. A., B&#250;zov&#225;, D., Liter&#225;kov&#225;, P., &#268;erven&#253;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+model+cyanobacterium+Synechocystis+sp:+PCC+6803+autotrophic+growth+in+a+flat-panel+photobioreactor.">Characterization of a model cyanobacterium Synechocystis sp: PCC 6803 autotrophic growth in a flat-panel photobioreactor.</a> Engineering in Life Sciences. 15 (1), (2015).</li><li>Zav&#345;el, T., O&#269;en&#225;&#353;ov&#225;, P., &#268;erven&#253;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+Synechocystis+sp.+PCC+6803+substrains+reveals+differences+in+sensitivity+to+abiotic+stress.">Phenotypic characterization of Synechocystis sp. PCC 6803 substrains reveals differences in sensitivity to abiotic stress.</a> PLoS One. 12 (12), e0189130 (2017).</li><li>Bennett, A., Bogorad, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+chromatic+adaption+in+a+filamentous+blue-green+alga.">Complementary chromatic adaption in a filamentous blue-green alga.</a> The Journal of Cell Biology. 58, 419-435 (1973).</li><li>L&#252;der, U. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+estimating+R-phycoerythrin+and+R-phycocyanin+contents+from+crude+aqueous+extracts+of+Porphyra+(Bangiales,+Rhodophyta).">An improved method for estimating R-phycoerythrin and R-phycocyanin contents from crude aqueous extracts of Porphyra (Bangiales, Rhodophyta).</a> Journal of Applied Phycology. 19 (2), 123-129 (2007).</li><li>Chung, Y. H., Park, Y. M., Moon, Y. J., Lee, E. M., Choi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photokinesis+of+Cyanobacterium+Synechocystis+sp.+PCC+6803.">Photokinesis of Cyanobacterium Synechocystis sp. PCC 6803.</a> Journal of Photoscience. 11 (3), 89-94 (2004).</li><li>Sun, L., Gault, P. M., Marler, H. 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. A., B&#250;zov&#225;, D., Liter&#225;kov&#225;, P., &#268;erven&#253;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+model+cyanobacterium+Synechocystis+sp.+PCC+6803+autotrophic+growth+in+a+flat-panel+photobioreactor.">Characterization of a model cyanobacterium Synechocystis sp. 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. S., 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=Effect+of+high+pH+on+growth+of+Synechocystis+sp.+PCC+6803+cultures+and+their+contamination+by+golden+algae+(Poterioochromonas+sp.).">Effect of high pH on growth of Synechocystis sp. PCC 6803 cultures and their contamination by golden algae (Poterioochromonas sp.).</a> Applied Microbiology and Biotechnology. 100 (3), 1333-1341 (2016).</li><li>Ishii, A., Hihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+AbrB-Like+Transcriptional+Regulator,+Sll0822,+Is+Essential+for+the+Activation+of+Nitrogen-Regulated+Genes+in+Synechocystis+sp.+PCC+6803.">An AbrB-Like Transcriptional Regulator, Sll0822, Is Essential for the Activation of Nitrogen-Regulated Genes in Synechocystis sp. PCC 6803.</a> Plant Physiology. 148 (1), 660-670 (2008).</li></ol>

Denne protokollen beskriver en enkel, rask og reproduserbar metode for kvantifisering av phycobiliprotein innhold i modellen cyanobacterium Synechocystis. Flere metoder for cellen homogenisering, protein utvinning og phycocyanin og allophycocyanin kvantifisering blir sammenliknet, og den endelige protokollen representerer en kombinasjon av optimal trinnene på hver enkelt prosedyre. Som representant, ble innholdet i phycobiliproteins kvantifisert i Synechocystis celler under økende lysintensiteten. Sel...

fPhycobiliproteins er vannløselige pigment-protein komplekser som representerer store deler av lys-fangst antenner i prokaryote Cyanobakterie (Cyanophyta) og flere eukaryote taxa (Glaucophyta, Rhodophyta , og Cryptophyta)1. De oppstår hovedsakelig som supramolecular komplekser kalt phycobilisomes, og de er vanligvis festet til overflaten av fotosynteseaktiviteten membraner på stromal side, med unntak av Cryptophyta, hvor phycobiliproteins er lokalisert i den thylakoid lumen2. Fire typer phycobiliproteins er identifisert oppdatert: core allophycocyanin og den eksterne phycocyanin, phycoerythrin og phycoerythrocyanin1. Som de viktigste lys-fangst kompleksene representerer phycobilisomes en av de avgjørende faktorene alger og Cyanobakterie masse kulturer produktivitet. Det har vist at phycobilisomes trunkering kan forbedre biomasse akkumulering under sterkt lys3. På den annen side, under beskjeden eller lav Irradians resulterte antenne trunkering i vekst og biomasse akkumulering reduksjon3,4. Phycobiliproteins brukes kommersielt som mat colorants, farmasi og tilsetningsstoffer, i kosmetisk industri, og fluorescens sonder programmer flowcytometri, fluorescerende immunanalyser og fluorescens mikroskopi5.
Denne protokollen fokuserer på kvantitative fastsettelse av phycobiliproteins i modellen cyanobacterium Synechocystis. Cyanobakterie er de tidligste oxygenic fotosynteseaktiviteten autotrophs; de har dannet jordens biosfære for mer enn 2,4 milliarder år6. De spiller en avgjørende rolle i globale biogeochemical sykluser av nitrogen, karbon, oksygen og andre elementer. Blant Cyanobakterie, en encellet belastning Synechocystis fikk en unik posisjon siden det var den første cyanobacterium med hele genomet sekvensert7,8, er det naturlig transformable av eksogene DNA9, og Det utfører stabil og relativt rask vekst10,11. I Synechocystis, antennen kjernekomponent, allophycocyanin, er forbundet med integrert membran proteiner og den tilknyttede phycocyanin ligger på thylakoid membran periferi.
Flere metoder for phycobiliprotein utvinning og kvantifisering sammenlignes i denne protokollen. Den endelige uttrekket prosedyren ble brukt til Synechocystisog andre Cyanobakterie stammer, inkludert Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira SP., og Nostoc sp., og det ble også brukt til Rødalger Porphyridium cruentum. Derfor må metoden utviklet i denne protokollen betraktes som en universell metode for phycobiliprotein utvinning. Selv om noen av metodene testet utvinning resulterte i høyere totale protein avkastning, her beskrevet utvinning prosedyre forutsatt den høyeste phycobiliprotein gir sammen med lavest innholdet av klorofyll en rest i den phycobiliprotein ekstrakt. Redusere innholdet av klorofyll en var avgjørende for riktig phycocyanin og allophycocyanin Spektrofotometri kvantifisering.
Phycobiliprotein absorpsjon spectra kan variere betydelig mellom ulike alger og Cyanobakterie arter12,13,14,15,16,17 og blant flere stammer av en enkelt Cyanobakterie slekten18. Derfor er bestemte bølgelengder og absorpsjon koeffisientene som brukes til fastsettelse av phycocyanin og allophycocyanin i Synechocystis ikke generelt gjelder for andre stammer. I tillegg inneholder ikke Synechocystis phycoerythrin og phycoerythrocyanin som finnes i noen andre alger og Cyanobakterie. For fastsettelse av phycobiliproteins i stammer enn Synechocystis, er det anbefalt å evaluere hver stamme Spektrofotometri ligninger individuelt.
Selv om protokollen inneholder to lengre trinn (over natten Frysetørring mobilnettet pellets og 1-timers protein utvinning), er den totalantallet phycobiliproteins kvantifiseringen lengre enn 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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR 80-Place Storage System</td>
			<td style="width:417px;">VWR International, Radnor, Pennsylvania, USA</td>
			<td>30128-282</td>
			<td>Holder for safe-lock tubes&#160;</td>
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			<td height="21" style="height:21px;">RAININ 100 &#181;l -1000 &#181;l&#160;</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>17014382</td>
			<td>Pipette</td>
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			<td height="21" style="height:21px;">GP-LTS-A-1000&#181;L-/F-768/8</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
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			<td height="21" style="height:21px;">Rotina 420R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td>4701</td>
			<td>Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes</td>
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			<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 enkel protokoll for kvantitative fastsettelse av phycobiliprotein innhold i modellen cyanobacterium Synechocystis. Phycobiliproteins er de viktigste komponentene i phycobilisomes, store lys-fangst antenner i Cyanobakterie og flere alger taxa. Phycobilisomes av Synechocystis inneholder to phycobiliproteins: phycocyanin og allophycocyanin. Denne protokollen beskriver en enkel, effektiv og pålitelig metode for kvantitative fastsettelse av både phycocyanin og allophycocyanin i denne modellen cyanobacterium. Vi sammenlignet flere metoder for phycobiliprotein utvinning og Spektrofotometri kvantifisering. Utvinning prosedyren som beskrevet i denne protokollen ble også brukt til andre Cyanobakterie belastninger Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., og Nostoc sp., samt om Rødalger Porphyridium cruentum. Men utryddelse koeffisientene til bestemte phycobiliproteins fra ulike taxa kan variere og, derfor anbefales det å validere Spektrofotometri kvantifisering metoden for hver enkelt belastning individuelt. Den krever lite tid og kan utføres i alle standard biovitenskap laboratorium siden det krever bare standard utstyr.

For de første metoden testene, Synechocystis ble kultivert som satsvise kulturer i Erlenmeyer flasker på en shaker i BG11 dyrking middels20 (supplert med 17 mM HEPES) ved 25 ° C, under et varmt hvitt lys med en intensitet av 50 µmol (fotoner) / (m 2·s) og med 1% CO2 culturing atmosfære. Under dyrking, kulturer var samplet safe-lock rør og sentrifugeres (15.000 x g laboratorium temperatur i 5 min), nedbryting ble forkas...

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

Her presenterer vi en protokoll for å finne kvantitativt phycobiliprotein innhold i cyanobacterium Synechocystis med en Spektrofotometri metode. Utvinning prosedyren ble også brukt til andre Cyanobakterie og alger stammer; men på grunn av variasjoner i pigment absorpsjon spectra er det nødvendig å teste hver stamme Spektrofotometri ligninger individuelt.

Spektrofotometri fastsettelse av Phycobiliprotein innhold 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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