Name: spectrophotometric determination of phycobiliprotein content in cyanobacterium synechocystis
Uploaded: 2018-09-11T13:30:00
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Protokol bir önceki yayın11kabul edilmiştir. T. desteklenen Milli Eğitim Bakanlığı, gençlik ve spor ulusal sürdürülebilirlik programı içinde Çek Cumhuriyeti tarafından Z., ö. bölüm ve J. Č. ben (NPU ben), sayı LO1415 verin. J. Č. Ayrıca GA CR, Grant sayı 18-24397S tarafından desteklenmiştir. Erişim cihazları ve diğer özellikler için Çek araştırma altyapı sistemleri biyoloji C4SYS (proje yok LM2015055) tarafından desteklenmiştir. M. A. S. Rus Bilim Vakfı [No 14-14-00904] bir hibe tarafından desteklenmiştir.

1. Siyanobakteriler ekimi

<ol><li>Synechocystis hücreleri Erlenmeyer şişe veya < 10 (17 mM HEPES10kullanarakÖrneğin,) pH korumak için arabelleğe alınan BG11 orta20 photobioreactors10,19 yetiştirmek. Not: Standart yetiştirme koşulları kontrollü bir sıcaklık gereklidir (genellikle, 30 ° C, en uygun sıcaklık 35 ° C olur)21, aydınlatma (tipik ola...

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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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Bu iletişim kuralı modeli cyanobacterium Synechocystisiçeriğinde phycobiliprotein miktar için basit, hızlı ve tekrarlanabilir yöntemi açıklar. Hücre homojenizasyon, protein çıkarma ve phycocyanin ve allophycocyanin miktar birkaç yöntem karşılaştırılır ve son iletişim kuralı tek her yordam en iyi adımlardan bir birleşimini temsil eder. Temsilcisi veri phycobiliproteins içeriğini ışık şiddeti artan altında Synechocystis hücrelerdeki sayısal. Labaratuar donanımları daha ...

fPhycobiliproteins prokaryotik Siyanobakteriler (Cyanophyta) ışık-hasat anten ve birçok ökaryotik takson (Glaucophyta, Rhodophyta ana bileşenlerini temsil suda çözünen pigment-protein kompleksleri vardır ve Cryptophyta)1. Bunlar esas supramolecular kompleksleri phycobilisomes denilen ortaya ve fotosentetik membran yüzeyine stromal tarafında, Cryptophytanereye phycobiliproteins lokalize dışında genellikle takıldıkları thylakoid lümen2. Phycobiliproteins dört tür tarihine kadar tespit edilmiştir: çekirdek allophycocyanin ve periferik phycocyanin, phycoerythrin ve phycoerythrocyanin1. Işık-hasat ana kompleksler, phycobilisomes yosun ve Siyanobakteriler kitle kültürü verimlilik çok önemli faktörlerden biri temsil eder. O phycobilisomes kesme biyokütle birikimi güçlü ışık3altında geliştirebilirsiniz kanıtlanmıştır. Öte yandan, mütevazı veya düşük olma altında büyüme oranları ve biyokütle birikimi azaltma3,4anten kesme sonuçlandı. Phycobiliproteins ticari olarak yiyecek renklendirici, eczacılık ve kozmetik sanayi, gıda katkı maddeleri ve floresan olarak kullanılan probları akış sitometresi, floresan uzun ve floresan mikroskopi5uygulamaları ile.
Bu iletişim kuralı modeli cyanobacterium Synechocystisphycobiliproteins kantitatif tayin odaklanır. Siyanobakteriler temel erken fotosentetik Ototrofların vardır; Onlar Dünya'nın biyosfer 2.4 milyar yıl6' dan daha fazla bilgi için oluşturmuşlardır. Onlar azot, karbon, oksijen ve diğer öğeleri global biyojeokimyasal döngüleri çok önemli bir rol oynamaktadır. Siyanobakteriler arasında tek hücreli zorlanma tüm genomu ile ilk cyanobacterium olduğu benzersiz bir konum elde Synechocystis 7,8sıralı, eksojen DNA9tarafından doğal olarak transformable ve nispeten hızlı ve istikrarlı büyüme10,11gerçekleştirir. Synechocystis, çekirdek anten bileşeni allophycocyanin, integral membran proteinlerinin ile ilişkili ve ekli phycocyanin thylakoid membran çevre üzerinde yer alır.
Phycobiliprotein ayıklama ve miktar için birkaç yöntem bu protokolde karşılaştırılır. Son çıkarma yordamı başarıyla Synechocystisyanı sıra Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira de dahil olmak üzere diğer Siyanobakteriler suşları için uygulandı SP.ve Nostoc sp. ve de başarıyla uygulanma kırmızı alg Porphyridium cruentum. Bu nedenle, bu protokol için geliştirilen Yöntem phycobiliprotein çıkarılması için evrensel bir yöntem olarak kabul edilebilir. Birkaç test ayıklama yöntemi daha yüksek toplam protein verimleri sonuçlandı olsa bile, klorofil bir kalıntı en düşük içeriği ile birlikte en yüksek phycobiliprotein verimleri sağlanan burada açıklanan çıkarma yordamı phycobiliprotein ayıklayın. Klorofil bir içeriğini azaltmak doğru phycocyanin ve allophycocyanin spektrofotometrik miktar için gerekli.
Phycobiliprotein emme spectra önemli ölçüde çeşitli yosun ve Siyanobakteriler türler12,13,14,15,16,17 arasında farklılık gösterir ve hatta bir tek Siyanobakteriler cins18çeşitli suşları arasında. Bu nedenle, belirli dalga boylarında ve phycocyanin ve Synechocystis allophycocyanin tespiti için kullanılan gibi emme katsayıları genellikle diğer suşları için uygun değildir. Ayrıca, Synechocystis phycoerythrin ve bazı diğer algler ve Siyanobakteriler bulunan phycoerythrocyanin içermiyor. Suşları Synechocystisdışında phycobiliproteins belirlenmesi amacıyla spektrofotometrik denklemler her zorlanma için ayrı ayrı değerlendirmeniz önerilir.
Protokol (hücresel granül ve 1 saatlik protein çıkarma gecede dağılması) iki uzun adımlar içerse de, phycobiliproteins miktar toplam çalışma zamanı 2 saatten daha uzun geldi.

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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;">GP-LTS-A-1000&#181;L-/F-768/8</td>
			<td>Mettler-Toledo, Columbus, Ohio, USA</td>
			<td>30389272</td>
			<td>Pipette tips</td>
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		<tr height="21">
			<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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		<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

Bu modeli cyanobacterium Synechocystisiçeriğinde phycobiliprotein kantitatif tayini için basit bir protokoldür. Phycobiliproteins phycobilisomes, büyük ışık-hasat anten Siyanobakteriler içinde en önemli bileşenleri ve birkaç yosun özellikleri vardır. Synechocystis phycobilisomes iki phycobiliproteins içerir: phycocyanin ve allophycocyanin. Bu iletişim kuralı, verimli, basit ve güvenilir yöntem phycocyanin ve allophycocyanin bu modeli cyanobacterium yılında niceliksel belirlenmesi için açıklar. Biz phycobiliprotein çıkarma ve spektrofotometrik miktar birkaç yöntem göre. Bu protokol için açıklandığı gibi çıkarma yordamı Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., gibi diğer Siyanobakteriler suşları için de başarıyla uygulandı ve Nostoc sp., de kırmızı alg Porphyridium cruentumolarak. Ancak, çeşitli özellikleri üzerinden belirli phycobiliproteins tükenme katsayıları farklı olabilir ve bu nedenle, tek tek tek her zorlanma için spektrofotometrik miktar yöntemi doğrulamak için tavsiye edilir. Protokol az zaman gerektirir ve yalnızca standart donanım gerektirir beri herhangi bir standart yaşam bilim laboratuarında gerçekleştirilebilir.

İlk yöntem sınavlara Erlenmeyer şişe üzerinde bir shaker kültürlerde toplu olarak ekili Synechocystis (17 mM HEPES ile desteklenmiş) BG11 ekimi orta-20 dakika 50 µmol (fotonlar) bir yoğunluk sıcak bir beyaz ışık altında 25 ° C / (m 2·s) ve kodlamayla atmosferde % 1 CO2 . Ekimi sırasında kültür güvenli kilit tüpler için örnek ve (15.000 x g 5 min için laboratuvar sıcaklığında) centrifuged, süper...

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

Burada, kantitatif spektrofotometrik yöntemle Synechocystis cyanobacterium phycobiliprotein içeriğini belirlemek için bir iletişim kuralı mevcut. Çıkarma yordamı diğer Siyanobakteriler ve yosun suşları için de başarıyla uygulandı; Ancak, pigment soğurma spektrumları farklılığı nedeniyle, tek tek her zorlanma için spektrofotometrik denklemler test gereklidir.

Cyanobacterium Synechocystis Phycobiliprotein içeriğinde spektrofotometrik tayini

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

Research

JoVE Journal

Biochemistry

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Etkili belirlenmesi tahlillerinde Peptit Agonistlerinin seri Seyreltme için Dağıtım Otomatik Akustik

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Fosforilasyon Saha Tespiti İçin Oligopeptide Rekabet Testi

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Determination of the Glycogen Content in Cyanobacteria

Siyanobakterlerde Glikojen İçeriğinin Belirlenmesi

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Yüksek işlem hacmi, mutlak nicel nokta kullanarak doku düzeyleri, seçili bir Protein içeriği tayini Blot Analizi (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

A Colorimetric Method for Measuring Iron Content in Plants

Demir içeriği tesislerinde ölçme kolorimetrik yöntemi

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Yüksek içeriği hedef nişan yapisan hücrelerdeki ölçmek için Imaging kullanarak

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

MicroRNA ile yerli lipoprotein parçacıkları zenginleştirme ve onların mutlak/bağıl microRNA Içerik ve onların hücresel transfer hızı sonraki belirlenmesi

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Sitosolion Glutatyon S-Transferazlarının Potansiyel Inhibitörleri için Spektrofotometrik Tarama

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Kitle Fotometrisi ile Antikor-Antijen Affinitesinin Hızlı Tayini

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

Çok Yönlü Bir Mikroakışkan Çipte Seri Kristalografi ile Bir Enzim:Substrat Kompleksinin Kristalizasyonu ve Yapısal Belirlenmesi

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...