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

The authors acknowledge Nordic Energy Research (AquaFEED, project no. 24), Innovationfonden Denmark (Pant Power, project no. 12-131844), and Villum Fonden (project no. 13363)

1. Preparation
<ol>
	<li>Cyanobacterial cultures
	<ol>
		<li>Grow Synechocystis sp. PCC 6803 at 30 &#176;C in liquid BG11 medium8, with a constant supply of air supplemented with 1% (v/v) CO2. Illuminate the cultures continuously with light at a photosynthetic photon flux density of 50 &#181;mol photon/m2/s.</li>
		<li>Grow Synechococcus sp. PCC 7002 in liquid A+ medium23 (BG11 medium can also be used), with a const...

<ol>
	<li>Chen, X., 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=The+Entner-Doudoroff+pathway+is+an+overlooked+glycolytic+route+in+cyanobacteria+and+plants.">The Entner-Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants.</a> Proc Natl Acad Sci USA. 113 (19), 5441-5446 (2016).</li><li>Yang, C., Hua, Q., Shimizu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+flux+analysis+in+Synechocystis+using+isotope+distribution+from+C-13-labeled+glucose.">Metabolic flux analysis in Synechocystis using isotope distribution from C-13-labeled glucose.</a> Metab Eng. 4 (3), 202-216 (2002).</li><li>Pelroy, R. A., Levine, G. A., Bassham, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+light-dark+CO2+fixation+and+glucose+assimilation+by+Aphanocapsa+6714.">Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714.</a> J Bacteriol. 128, 633-643 (1976).</li><li>You, L., Berla, B., He, L., Pakrasi, H. B., Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-MFA+delineates+the+photomixotrophic+metabolism+of+Synechocystis.+sp.+PCC+6803+under+light-+and+carbon-sufficient+conditions.">13C-MFA delineates the photomixotrophic metabolism of Synechocystis. sp. PCC 6803 under light- and carbon-sufficient conditions.</a> Biotechnol J. 9, 684-692 (2014).</li><li>Jacobsen, J. H., Frigaard, N. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+photosynthetic+mannitol+biosynthesis+from+CO2+in+a+cyanobacterium.">Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium.</a> Metab Eng. 21, 60-70 (2014).</li><li>M&#246;llers, K. B., Cannella, D., J&#248;rgensen, H., Frigaard, N. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacterial+biomass+as+carbohydrate+and+nutrient+feedstock+for+bioethanol+production+by+yeast+fermentation.">Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation.</a> Biotechnol Biofuels. 7, 64 (2014).</li><li>Angermayr, S. A., 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=Culturing+Synechocystis.+sp.+strain+PCC+6803+with+N2+and+CO+2+in+a+diel+regime+reveals+multiphase+glycogen+dynamics+with+low+maintenance+costs.">Culturing Synechocystis. sp. strain PCC 6803 with N2 and CO 2 in a diel regime reveals multiphase glycogen dynamics with low maintenance costs.</a> Appl Environ Microbiol. 82, 4180-4189 (2016).</li><li>de Porcellinis, A. 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=The+Non-coding+RNA+Ncr0700/PmgR1+is+required+for+photomixotrophic+growth+and+the+regulation+of+glycogen+accumulation+in+the+cyanobacterium+Synechocystis+sp.+PCC+6803.">The Non-coding RNA Ncr0700/PmgR1 is required for photomixotrophic growth and the regulation of glycogen accumulation in the cyanobacterium Synechocystis sp. PCC 6803.</a> Plant Cell Physiol. 57 (10), 2091-2103 (2016).</li><li>Sakuragi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Tocopherol+plays+a+role+in+photosynthesis+and+macronutrient+homeostasis+of+the+cyanobacterium+Synechocystis+sp.+PCC+6803+that+is+independent+of+its+antioxidant+function.">alpha-Tocopherol plays a role in photosynthesis and macronutrient homeostasis of the cyanobacterium Synechocystis sp. PCC 6803 that is independent of its antioxidant function.</a> Plant Physiol. 141, 508-521 (2006).</li><li>Osanai, 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=Positive+regulation+of+sugar+catabolic+pathways+in+the+cyanobacterium+Synechocystis.+sp.+PCC+6803+by+the+group+2+sigma+factor+sigE.">Positive regulation of sugar catabolic pathways in the cyanobacterium Synechocystis. sp. PCC 6803 by the group 2 sigma factor sigE.</a> J Biol Chem. 280, 30653-30659 (2005).</li><li>Yamauchi, Y., Kaniya, Y., Kaneko, Y., 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=Physiological+roles+of+the+cyAbrB+transcriptional+regulator+pair+Sll0822+and+Sll0359+in+Synechocystis+sp.+strain+PCC+6803.">Physiological roles of the cyAbrB transcriptional regulator pair Sll0822 and Sll0359 in Synechocystis sp. strain PCC 6803.</a> J Bacteriol. 193, 3702-3709 (2011).</li><li>Dubois, M., Gilles, K., Hamilton, J., Rebers, P., Smith, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+method+for+determination+of+sugars+and+related+substances.">Colorimetric method for determination of sugars and related substances.</a> Anal Chem. 28, 350-356 (1956).</li><li>Osanai, 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=Genetic+engineering+of+group+2+{sigma}+factor+SigE+widely+activates+expressions+of+sugar+catabolic+genes+in+Synechocystis+species+PCC+6803.">Genetic engineering of group 2 {sigma} factor SigE widely activates expressions of sugar catabolic genes in Synechocystis species PCC 6803.</a> J Biol Chem. 286, 30962-30971 (2011).</li><li>Miao, X., Wu, Q., Wu, G., Zhao, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+accumulation+in+salt-stressed+cells+of+agp+gene+deletion-mutant+in+cyanobacterium+Synechocystis+sp.+PCC+6803.">Sucrose accumulation in salt-stressed cells of agp gene deletion-mutant in cyanobacterium Synechocystis sp. PCC 6803.</a> FEMS Microbiol Letters. 218, 71-77 (2003).</li><li>Hagemann, M., Erdmann, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+pathway+of+glucosylglycerol+synthesis+in+the+cyanobacterium+Synechocystis+sp.+PCC+6803.">Activation and pathway of glucosylglycerol synthesis in the cyanobacterium Synechocystis sp. PCC 6803.</a> Microbiology. 140, 1427-1431 (1994).</li><li>Nobles, D. R., Romanovicz, D. K., Brown, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+in+cyanobacteria.+Origin+of+vascular+plant+cellulose+synthase.">Cellulose in cyanobacteria. Origin of vascular plant cellulose synthase.</a> Plant Physiol. 127, 529-542 (2001).</li><li>Zhao, 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=High-yield+production+of+extracellular+type-I+cellulose+by+the+cyanobacterium+Synechococcus+sp.+PCC+7002.">High-yield production of extracellular type-I cellulose by the cyanobacterium Synechococcus sp. PCC 7002.</a> Cell Discovery. 1, 15004 (2015).</li><li>Kawano, Y., 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=Cellulose+accumulation+and+a+cellulose+synthase+gene+are+responsible+for+cell+aggregation+in+the+cyanobacterium+Thermosynechococcus+vulcanus+RKN.">Cellulose accumulation and a cellulose synthase gene are responsible for cell aggregation in the cyanobacterium Thermosynechococcus vulcanus RKN.</a> Plant Cell Physiol. 52, 957-966 (2011).</li><li>Angermayr, S. A., Gorchs Rovira, A., Hellingwerf, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+cyanobacteria+for+the+synthesis+of+commodity+products.">Metabolic engineering of cyanobacteria for the synthesis of commodity products.</a> Trends Biotechnol. 33, 352-361 (2015).</li><li>Zhang, B., Dhital, S., Gidley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+and+antagonistic+effects+of+&#945;-amylase+and+amyloglucosidase+on+starch+digestion.">Synergistic and antagonistic effects of &#945;-amylase and amyloglucosidase on starch digestion.</a> Biomacromolecules. 14, 1945-1954 (2013).</li><li>Harholt, 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=ARABINAN+DEFICIENT+1+is+a+putative+arabinosyltransferase+involved+in+biosynthesis+of+pectic+arabinan+in+Arabidopsis.">ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in biosynthesis of pectic arabinan in Arabidopsis.</a> Plant Physiol. 140, 49-58 (2006).</li><li>Fernando, C. D., Soysa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+enzymatic+colorimetric+assay+for+determination+of+hydrogen+peroxide+(H2O2)+scavenging+activity+of+plant+extracts.">Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts.</a> MethodsX. 2, 283-291 (2015).</li><li>Jacobsen, J. H., Rosgaard, L., Sakuragi, Y., Frigaard, N. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+plasmid+construction+for+generation+of+knock-out+mutants+in+cyanobacteria:+studies+of+glycogen+metabolism+in+Synechococcus+sp+PCC+7002.">One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp PCC 7002.</a> Photosynth Res. 107 (2), 215-221 (2011).</li><li>Lichtenthaler, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophylls+and+carotenoids:+Pigments+of+photosynthetic+biomembranes.">Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes.</a> Methods Enzymol. 148, 350-382 (1987).</li><li>L&#243;pez, C. V. G., del Carmen Cer&#243;n Garc&#237;a, M., Fern&#225;ndez, F. G. A., Bustos, C. S., Chisti, Y., Sevilla, J. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurements+of+microalgal+and+cyanobacterial+biomass.">Protein measurements of microalgal and cyanobacterial biomass.</a> Bioresource Technol. 101, 7587-7591 (2010).</li><li>Hasunuma, 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=Dynamic+metabolic+profiling+of+cyanobacterial+glycogen+biosynthesis+under+conditions+of+nitrate+depletion.">Dynamic metabolic profiling of cyanobacterial glycogen biosynthesis under conditions of nitrate depletion.</a> J Exp Bot. 64, 2943-2954 (2013).</li><li>D&#237;az-Troya, S., L&#243;pez-Maury, L., S&#225;nchez-Riego, A. M., Rold&#225;n, M., Florencio, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+regulation+of+glycogen+biosynthesis+in+the+cyanobacterium+Synechocystis+sp.+PCC+6803:+Analysis+of+the+AGP+and+glycogen+synthases.">Redox regulation of glycogen biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803: Analysis of the AGP and glycogen synthases.</a> Molecular Plant. 7, 87-100 (2014).</li><li>Parrott, L. M., Slater, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA,+RNA+and+protein+composition+of+the+cyanobacterium+Anacystis+nidulans+grown+in+light-+and+carbon+dioxide-limited+chemostats.">The DNA, RNA and protein composition of the cyanobacterium Anacystis nidulans grown in light- and carbon dioxide-limited chemostats.</a> Arch Microbiol. 127, 53-58 (1980).</li><li>Hihara, Y., Kamei, A., Kanehisa, M., Kaplan, A., Ikeuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+microarray+analysis+of+cyanobacterial+gene+expression+during+acclimation+to+high+light.">DNA microarray analysis of cyanobacterial gene expression during acclimation to high light.</a> Plant Cell. 13, 793-806 (2001).</li></ol>

Critical steps within the protocol are glycogen precipitation and resuspension. After centrifugation following ethanol precipitation, glycogen forms a translucent pellet that loosely adheres to the walls of the microcentrifuge tubes. Therefore, when removing the supernatant, special attention needs to be given so as not to remove the pellet. The glycogen pellet is sticky, and solubilization can be difficult if it dries out. Note that the complete solubilization of the glycogen pellet is important because incomplete solub...

Cyanobacteria accumulate glycogen as the major carbohydrate store of carbon from CO2 fixed in light through photosynthesis. Glycogen is a glycan consisting of linear &#945;-1,4-linked glucan with branches created by &#945;-1,6-linked glucosyl linkages. Glycogen biosynthesis in cyanobacteria starts with the conversion of glucose-6-phosphate into ADP-glucose through the sequential action of phosphoglucomutase and ADP-glucose pyrophosphorylase. The glucose moiety in ADP-glucose is transferred to the non-reducing end of the &#945;-1,4-glucan backbone of glycogen by one or more glycogen synthases (GlgA). Subsequently, a branching enzymes introduce the &#945;-1,6-linked glucosyl linkage, which is further extended to generate the glycogen particle. In the dark, glycogen is broken down by glycogen phosphorylase, glycogen debranching enzymes, &#945;-glucanotransferase, and malto-dextrin phosphorylase into phosphorylated glucose and free glucose. These feed into catabolic pathways, including the oxidative pentose phosphate pathway, the Embden-Meyerhof-Parnas pathway (glycolysis), and the Entner-Doudoroff pathway1,2,3,4.
Glycogen metabolism in cyanobacteria has garnered increasing interest in recent years because of the potential for cyanobacteria to develop into microbial cell factories driven by sunlight to produce chemicals and fuels. Glycogen metabolism could be modified to increase the yield of the products, because glycogen is the largest flexible carbon pool in these bacteria. An example is the cyanobacterium Synechococcus sp. PCC 7002, which has been genetically engineered to produce mannitol; the genetic disruption of glycogen synthesis increases the mannitol yield 3-fold5. Another example is the production of bioethanol from glycogen-loaded wildtype Synechococcus sp. PCC 70026. The wildtype cell glycogen content may be up to 60% of the dry weight of the cell during nitrogen starvation6.
Our understanding of glycogen metabolism and regulation has also expanded in recent years. While glycogen is known to accumulate in the light and to be catabolized in the dark, detailed kinetics of glycogen metabolism during the diel cycle was only recently revealed in Synechocystis sp. PCC 68037. Moreover, several genes affecting the accumulation of glycogen have been identified. A notable example is the discovery that the putative histidine kinase PmgA and the non-coding RNA PmgR1 form a regulatory cascade and control the accumulation of glycogen. Interestingly, the pmgA and pmgR1 deletion mutants accumulate twice as much glycogen as the wildtype strain of Synechocystis sp. PCC 68038,9. Other regulatory elements are also known to affect the accumulation of glycogen, including the alternative sigma factor E and the transcriptional factor CyAbrB210,11.
As interest in glycogen regulation and metabolism grow, a detailed protocol describing the determination of the glycogen content is warranted. Several methods are used in the literature. Acid hydrolysis followed by the determination of the monosaccharide content through high-pressure anion exchange liquid chromatography coupled with a pulsed amperometric detector or spectrometric determination following treatments with acid and phenol are widely used methods to approximate the glycogen content9,10,12,13. However, a high-pressure anion exchange liquid chromatographic instrument is very expensive and does not discriminate glucose derived from glycogen from that derived from other glucose-containing glycoconjugates, such as sucrose14, glucosylglycerol15, and cellulose16,17,18, which are known to accumulate in some cyanobacterial species. The acid-phenol method can be performed using standard laboratory equipment. However, it uses highly toxic reagents and does not distinguish glucose derived from different glycoconjugates, nor does it distinguish glucose from other monosaccharides that constitute cellular materials, such as glycolipids, lipopolysaccharides, and extracellular matrices12. Notably, the hot acid-phenol assay is often used for the determination of total carbohydrate content rather than for the specific determination of glucose content12. Enzymatic hydrolysis of glycogen to glucose by &#945;-amyloglucosidase followed by the detection of glucose through an enzyme-coupled assay generates a colorimetric readout that is highly sensitive and specific to glucose derived from glycogen. The specificity can be enhanced further with the preferential precipitation of glycogen from cell lysates by ethanol5,8,19.
Here, we describe a detailed protocol for an enzyme-based assay of the glycogen content in two of the most widely studied cyanobacterial species, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, in the wildtype and mutant strains. In order to ensure efficient hydrolysis, a cocktail of &#945;-amylase and &#945;-amyloglucosidase is used8. The endo-acting &#945;-amylase hydrolyzes the &#945;-1,4-linkages in various glucans into dextrins, which are further hydrolyzed to glucose by exo-acting &#945;-amyloglucosidase20. The synergistic effects of these enzymes are well known, and these enzymes are routinely used for the selective hydrolysis of starch, which is an &#945;-linked glucan like glycogen, without affecting other glycoconjugants, such as cellulose, in the plant biomass21. The released glucose is quantitatively detected following an enzyme-coupled assay consisting of glucose oxidase-which catalyzes the reduction of oxygen to hydrogen peroxide and the oxidation of glucose to a lactone-and peroxidase-which produces a pink-colored quinoneimine dye from hydrogen peroxide, a phenolic compound, and 4-aminoantipyrine22.

<table border="0" cellpadding="0" cellspacing="0" width="784">
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	<tbody>
		<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>
		</tr>
		<tr height="21">
			<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

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

10 mL of wildtype Synechocystis sp. PCC 6803 were grown under photoautotrophic conditions until the OD730nm value reached approximately 0.8. The cells were harvested and resuspended in 50 mM Tris-HCl, pH 8. The OD730nm value was adjusted to 2-3. The glycogen content was analyzed following the protocol described above. The glycogen content per the OD730nm was 13 &#177; 1.8 &#181;g/mL/OD730nm (N = 12). The glycogen content relati...

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Determination of the Glycogen Content in Cyanobacteria

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

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

The overall goal of this procedure is to determine the glycogen content in cyanobacteria using an enzyme-based selective hydrolysis and assay. This method can help answer a key question in the cyanobacteria field, such as physiology, molecular genetics, and bioengineering of different cyanobacteria strain under investigation. The main advantage of this technique is that it's adapted to a small scale, it's easy to perform, and highly sensitive and specific to glycogen.Though this method can provide insight into cyanobacteria, it can also be applied to other microorganisms that accumulate glycogen or starch, such as E.Coli, yeast, microalgae, and various heterotrophic and phototrophic microorganisms. Begin this protocol with preparation of cyanobacterial cultures as described in the text protocol. Transfer one milliliter of cyanobacterial culture or cell suspension to a 1.5 milliliter tube.Then centrifuge at 6, 000 times g and four degrees Celsius for 10 minutes. Discard the supernatant before resuspending the pellet in one milliliter of 50 millimolar Tris-HCL pH eight. Centrifuge the resuspended pellet as before.Discard the supernatant, and resuspend the cell pellet a second time in the Tris-HCL buffer. Centrifuge the tubes as before and discard the supernatant. Then thoroughly resuspend the pellet in 500 microliters of 50 millimolar Tris-HCL buffer pH eight.It is crucial to have the pellet well-suspended for an efficient lysis. Keep the resuspension in ice. Lyse the resuspended cells at four degrees Celsius by 30 cycles of ultrasonication with each cycle consisting of 30 seconds at a frequency of 20 kilohertz with a maximum amplitude, followed by 90 seconds without.Centrifuge the tube containing the lysate at 6, 000 times g and four degrees Celsius for 10 minutes. Following centrifugation, the pellet should be mainly large cell debris, and the supernatant is used for further analysis. At this point, determine the protein concentration by using the protein assay kit.Remove chlorophyll a from the cell lysate by mixing 900 microliters of ethanol and 100 microliters of the obtained supernatant in a 1.5 milliliter screw cap tube. After closing the cap, heat the tube at 90 degrees Celsius for 10 minutes, using a standard laboratory heating block. Then, incubate the tube in ice for 30 minutes.Next, centrifuge the tube at 20, 000 times g and four degrees Celsius for 30 minutes. Following centrifugation, carefully remove the supernatant. The pellet contains glycogen.Lightly dry the pellet in air to remove excess ethanol. Do not excessively dry the pellet to avoid difficult dissolving it. Measure the absorbance at 666 nanometers of the obtained supernatant to determine the chlorophyll a content.The value can be used to normalize the glycogen content. Dissolve the pellet in 100 microliters of 50 millimolar sodium acetate pH five. Mix these materials well, using a vortex.Mixing by pipetting is not recommended because the mixture is viscous. Also add 50 microliters of eight units per milliliter amyloglucosidase and 50 microliters of two units per milliliter of alpha-amylase. Next, incubate the mixture at 60 degrees Celsius in a heating block for two hours to enable the digestion of glycogen into glucose molecules.Following incubation, centrifuge the sample at 20, 000 times g for five minutes and transfer the supernatant to a new 1.5 milliliter tube. Measure the concentration of glucose in the supernatant following enzymatic hydrolysis, using the GOD-POD reagent. Transfer 100 microliters of the supernatant from the glycogen precipitation step to a well in a 96 well plate.As a negative control, use 100 microliters of 50 millimolar sodium acetate pH five. For generating the calibration curve, also measure the glucose standard solutions. Add 150 microliters of GOD-POD reagent to each sample and quickly mix by pipetting.Incubate the plate at 25 degrees Celsius for 30 minutes. Then, record the absorbance value at 510 nanometers, using a plate reader. Calculate the amount of glycogen as the glucose equivalent, using a calibration curve obtained from the glucose standards.The glycogen contents in synechocystis are shown here. The two mutant strains, delta pmg A and delta pmg R, which are known to hyperaccumulate glycogen, were compared to the wild type strain. As expected, the mutant strains showed elevated levels of glycogen.Conversely, a mutant that lacks glycogen synthase did not accumulate glycogen. The strains used here have been engineered to produce mannitol. Notably, the glycogen synthase mutant produced more mannitol than the control strain, which suggests that the carbohydrate synthesized by photosynthesis is redirected to mannitol in the mutant strain lacking the ability to synthesize glycogen.After watching this video, you should be able to quantitatively determine the glycogen content in cyanobacteria in five hours. When performing this procedure, it's important to remember to thoroughly resuspend the cells and solubilize glycogen pellets to obtain reliable results. Following this procedure, other methods like metabolic enzyme activity assays can be performed using the remaining cell lysates in order to answer additional questions, like how carbohydrate metabolism is regulated in cyanobacteria.

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