Name: determination of the glycogen content in cyanobacteria
Uploaded: 2017-07-17T15:00:00
Description: Watch this Scientific Journal Video about Determination of the Glycogen Content in Cyanobacteria at JoVE.com

作者承认北欧能源研究（AquaFEED，第24号项目），丹麦Innovationfonden（Pant Power，项目编号12-131844）和Villum Fonden（项目编号13363）

准备<ol><li> 蓝藻培养 <ol><li>生长集胞藻PCC 6803在30℃下在液体BG11培养基8中 ，具有补充有1％（v / v）CO 2的恒定空气供应。在光合光子通量密度为50μmol/ m 2 / s的条件下用光照亮培养物。 </li><li>生长聚球藻在液体A +介质23 （也可以使用BG11培养基）中的PCC 7002，补充有1％（v / v）CO 2的恒定空气供应。温度应为37°C?...

<ol>
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方案中的关键步骤是糖原沉淀和再悬浮。在乙醇沉淀后离心后，糖原形成半透明的颗粒，其松散地粘附到微量离心管的壁上。因此，当除去上清液时，需要特别注意不要去除颗粒。糖原颗粒是粘性的，并且如果干燥，则溶解变得困难。注意，糖原颗粒的完全溶解是重要的，因为不完全溶解将导致低效的酶消化，因此将在技术重复之间产生大的变化。在通过涡旋溶解之前应用超声处理可以有助于该?...

蓝细菌积累糖原作为通过光合作用固定在光中的来自CO 2的碳的主要碳水化合物储存。糖原是由线性α-1,4-连接葡聚糖组成的聚糖，其具有由α-1,6-连接的葡糖基键连接的分支。蓝细菌中的糖原生物合成开始于葡萄糖-6-磷酸转化为ADP-葡萄糖，通过磷酸果糖转移酶和ADP-葡萄糖焦磷酸化酶的顺序作用。 ADP-葡萄糖中的葡萄糖部分通过一种或多种糖原合成酶（GlgA）转移到糖原的α-1,4-葡聚糖骨架的非还原末端。随后，分支酶引入α-1,6-连接的葡糖基键，其进一步延伸以产生糖原颗粒。在黑暗中，糖原被糖原磷酸化酶，糖原脱支酶，α-葡聚糖转移酶和麦芽糊精磷酸化酶分解成磷酸化葡萄糖和游离葡萄糖。这些feed intø分解代谢途径，包括氧化戊糖磷酸途径，Embden-迈耶霍夫-Parnas途径（糖酵解），和ED途径1，2，3，4。 蓝藻中的糖原代谢近年来引起越来越多的兴趣，因为蓝藻潜力发展成由阳光驱动的微生物细胞工厂生产化学品和燃料。由于糖原是这些细菌中最大的柔性碳库，因此可以改变糖原代谢以增加产品的产量。一个例子是蓝细菌聚球藻PCC 7002已被基因工程化生产甘露醇;糖原合成的遗传破坏增加甘露醇产量3倍5 。另一个例子是从加载糖原的野猪生产生物乙醇ype Synechococcus sp。 PCC 7002 6 。野生型细胞的糖原含量可高达细胞的干重的60％的氮饥饿6中。 我们对糖原代谢和调节的理解近年来也有所扩大。虽然糖原已知在光​​中积聚并在黑暗中分解代谢，但在Diel循环中糖原代谢的详细动力学最近才在Synechocystis sp中显现。 PCC 6803 7 。此外，已经鉴定了几种影响糖原积累的基因。一个显着的例子是发现推定的组氨酸激酶PmgA和非编码RNA PmgR1形成调节级联并控制糖原的积累。有趣的是， pmgA和pmgR1缺失突变体与Synechocystis sp。的野生型菌株相比，积累了两倍的糖原。 PCC 68038，9。其他调控元件也已知会影响糖原的积累，包括替代σ因子E和转录因子CyAbrB2 10，11。 由于糖原调节和代谢的兴趣增长，需要一个描述糖原含量测定的详细方案。在文献中使用了几种方法。酸水解然后通过加上一个脉冲电流检测器或在用酸和酚处理光谱法测定高压阴离子交换液相色谱单糖含量的测定被广泛使用的方法来近似糖原含量9，10，12，13。然而，高压阴离子交换液相色谱仪C类仪器是非常昂贵的，并且不区分糖原从来自其它含葡萄糖的糖缀合物，例如蔗糖14，glucosylglycerol 15，和纤维素16，17，18，其已知在一些蓝藻物种积累衍生的葡萄糖。酸 - 酚法可以使用标准实验室设备进行。然而，它使用高毒性试剂，并且不区分来自不同糖缀合物的葡萄糖，也不区分葡萄糖与构成细胞材料的其它单糖，如糖脂，脂多糖和细胞外基质12 。值得注意的是，热酸-苯酚测定经常用于总碳水化合物含量，而不是的葡萄糖含量12的具体确定的确定。酶法通过α-淀粉葡萄糖苷酶将糖原溶解成葡萄糖，然后通过酶偶联测定法检测葡萄糖，产生对来自糖原的葡萄糖高度敏感和特异性的比色读数。特异性可以从细胞裂解物糖原的优先沉淀进一步用乙醇5，8，19来增强。 在这里，我们描述了两个最广泛研究的蓝藻种类Synechocystis sp。的糖基含量的基于酶的测定的详细方案。 PCC 6803和聚球藻PCC 7002，在野生型和突变株中。为了确保有效水解，α淀粉酶和α-淀粉葡萄糖苷酶的混合物被用于8。内效α-淀粉酶将各种葡聚糖中的α-1,4-键水解成糊精，进一步水解为o葡萄糖通过外效α-淀粉葡萄糖苷酶20 。这些酶的协同效应是众所周知的，并且这些酶通常用于选择性水解淀粉，其是α-连接葡聚糖样糖原，而不影响植物生物质21中的其它糖缀合物，例如纤维素。在葡萄糖氧化酶（其催化氧还原为过氧化氢）和葡萄糖氧化成内酯和过氧化物酶（其由过氧化氢产生粉红色醌亚胺染料）组成的酶偶联测定后，定量检测释放的葡萄糖，酚类化合物和4-氨基安替比林22 。

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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">QSonica Sonicators Q700</td>
			<td style="width:219px;">Qsonica, LLC</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;">QSonica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpectraMax 190 Microplate Reader&#160;</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:119px;">NA</td>
			<td>Eliza plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bullet Blender Storm</td>
			<td style="width:219px;">Next Advance</td>
			<td style="width:119px;">BBY24M-CE</td>
			<td>Beads beater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrospec 3100 pro UV/Visible Spectrophotometer</td>
			<td style="width:219px;">Amersham Biosciences</td>
			<td style="width:119px;">NA</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td>Buffer</td>
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			<td height="21" style="height:21px;width:280px;">HCl</td>
			<td style="width:219px;">Merck</td>
			<td style="width:119px;">1-00317</td>
			<td>pH adjutment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">32319</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amyloglycosidase (Rhizopus sp.)</td>
			<td style="width:219px;">Megazyme</td>
			<td style="width:119px;">E-AMGPU</td>
			<td>Enzyme for glycogen depolymerization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Amylase, thermostable (Bacillus licheniformis)</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3176</td>
			<td>Enzyme for glycogen depolymerization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:219px;">Merch</td>
			<td style="width:119px;">8337</td>
			<td>Standard for the glucose assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce BCA Protein assay kit&#160;</td>
			<td>Thermo Fisher scientific</td>
			<td style="width:119px;">23225</td>
			<td>For determination of protein concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Aluminum drying trays, disposable</td>
			<td style="width:219px;">VWR</td>
			<td style="width:119px;">611-1362</td>
			<td>For determination of cell dry weights</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">D-Glucose assay kit (GODPOD format)</td>
			<td style="width:219px;">Megazyme</td>
			<td style="width:119px;">K-GLUC</td>
			<td>For determination of glucose concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Zirconium oxide breads, 0.15 mm</td>
			<td style="width:219px;">Next Advance</td>
			<td style="width:119px;">ZrOB015</td>
			<td>Beads for cell lysis in a Bullet Blendar Storm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">RINO tubes</td>
			<td style="width:219px;">Next Advance</td>
			<td style="width:119px;">NA</td>
			<td>Tubes for cell lysis in a Bullet Blendar Storm</td>
		</tr>
	</tbody>
</table>

determination of the glycogen content in cyanobacteria

蓝藻在光合作用过程中积累糖原作为主要的细胞内碳和能量储存。研究的最新进展突出表明糖原代谢的复杂机制，包括生物合成和分解代谢的diel循环，氧化还原调节和非编码RNA的参与。同时，正在努力将碳从糖原转移到转基因工程蓝细菌中的所需产品，以提高产品产量。用几种方法测定蓝细菌中的糖原含量，具有不同的准确性和技术复杂性。在这里，我们提供了一个详细的协议，可以确定可以在标准生命科学实验室进行的蓝细菌中的糖原含量。该方案需要从细胞裂解液中糖原的选择性沉淀和糖原的酶促解聚以产生葡萄糖单体，其由葡萄糖酪氨酸过氧化物酶（GOD-POD）酶偶联测定。该方法已应用于集胞藻（Synechocystis sp。 PCC 6803和聚球藻PCC 7002，两种广泛应用于代谢工程的蓝藻种类。此外，该方法成功地显示了调节元件或糖原生物合成基因中的野生型和突变体之间的糖原含量的差异。

10 mL野生型集胞藻PCC 6803在光自养条件下生长，直到OD 730nm值达到约0.8。收集细胞并重悬于50mM Tris-HCl，pH8中。将OD 730nm值调节至2-3。按照上述方案分析糖原含量。每个OD 730nm的糖原含量为13±1.8μg/ mL / OD 730nm （ N = 12）。糖蛋白含量相对于蛋白质含量为0.24±0.03μg/μg（ N = 12），相对于叶绿素a含量的糖原含量为5.7±0.6μ...

Watch this Scientific Journal Video about Determination of the Glycogen Content in Cyanobacteria at JoVE.com

Determination of the Glycogen Content in Cyanobacteria

在这里，我们提出了一个可靠和容易的测定来测量蓝细菌细胞中的糖原含量。该过程需要沉淀，可选择解聚和葡萄糖残留的检测。该方法适用于野生型和遗传工程菌株，可促进蓝细菌的代谢工程。

测定蓝细菌中的糖原含量

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

Research

JoVE Journal

Biochemistry

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

自动声胶肽受体激动剂的效价测定测定的连续稀释

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

科研

生物化学

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

高亲和力抗体 - 抗原的结合动力学的测定，使用四个生物传感器平台

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

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

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

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

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

真菌病原体液体荧光恢复法测定脂质指数黑穗病玉米

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

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

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

分光光度法测定蓝藻Synechocystis中的蛋白含量

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

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

利用高含量成像对增强细胞中的目标参与进行量化

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

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

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

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

实现蛋白质快速光化学氧化的实时补偿，用于确定蛋白质地形变化

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...