Name: extraction of cofactor f420 for analysis of polyglutamate tail length from methanogenic pure cultures and environmental samples
Uploaded: 2021-10-14T14:00:00
Description: Watch this Scientific Journal Video about Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples at JoVE.com

我们非常感谢Colin Jackson教授对纯化辅因子F420的支持。这项研究得到了蒂罗尔科学基金（TWF）和因斯布鲁克大学（Publikationsfonds）的支持。我们非常感谢GPS，HK，SB，GG和HB的支持。

注意：辅因子F420 的提取和分析是一个三步过程，包括样品裂解，通过固相萃取（SPE）进行辅因子预纯，以及通过离子配对RP-HPLC（IP-RP-HPLC）和荧光检测进行辅因子检测。在开始之前，按照 表1中所述准备材料和试剂。
1. 样品裂解
<ol><li>将多达 5 g 样品加入适当的试管（例如，50 mL 锥形管）。</li>
	<li>向样品中加入5mL裂解缓冲液（2x储备溶液， 表1</st...

<ol>
	<li>Greening, 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=Physiology,+biochemistry,+and+applications+of+F420-+and+Fo-dependent+redox+reactions.">Physiology, biochemistry, and applications of F420- and Fo-dependent redox reactions.</a> Microbiology and Molecular Biology Reviews: MMBR. 80 (2), 451-493 (2016).</li><li>Bashiri, G., 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=A+revised+biosynthetic+pathway+for+the+cofactor+F420+in+prokaryotes.">A revised biosynthetic pathway for the cofactor F420 in prokaryotes.</a> Nature Communications. 10 (1), 451 (2019).</li><li>Grinter, R., Greening, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofactor+F420:+an+expanded+view+of+its+distribution,+biosynthesis,+and+roles+in+bacteria+and+archaea.">Cofactor F420: an expanded view of its distribution, biosynthesis, and roles in bacteria and archaea.</a> FEMS Microbiology Reviews. , (2021).</li><li>Eirich, L. D., Vogels, G. D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposed+structure+for+coenzyme+F+420+from+methanobacterium.">Proposed structure for coenzyme F 420 from methanobacterium.</a> Biochemistry. 17 (22), 4583-4593 (1978).</li><li>Ney, B., 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+methanogenic+redox+cofactor+F420+is+widely+synthesized+by+aerobic+soil+bacteria.">The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.</a> The ISME Journal. 11 (1), 125-137 (2017).</li><li>Braga, D., 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=Metabolic+pathway+rerouting+in+Paraburkholderia+rhizoxinica+evolved+long-overlooked+derivatives+of+coenzyme+F420.">Metabolic pathway rerouting in Paraburkholderia rhizoxinica evolved long-overlooked derivatives of coenzyme F420.</a> ACS Chemical Biology. 14 (9), 2088-2094 (2019).</li><li>Eirich, L. D., Vogels, G. D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+coenzyme+F420+and+properties+of+its+hydrolytic+fragments.">Distribution of coenzyme F420 and properties of its hydrolytic fragments.</a> Journal of Bacteriology. 140 (1), 20-27 (1979).</li><li>Michaelis, W., 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=Microbial+reefs+in+the+Black+Sea+fueled+by+anaerobic+oxidation+of+methane.">Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane.</a> Science. 297 (5583), 1013-1015 (2002).</li><li>Knittel, K., L&#246;sekann, T., Boetius, A., Kort, R., Amann, R. <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+distribution+of+methanotrophic+archaea+at+cold+seeps.">Diversity and distribution of methanotrophic archaea at cold seeps.</a> Applied and Environmental Microbiology. 71 (1), 467-479 (2005).</li><li>Lin, X. -. L., White, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+Coenzyme+F420+and+Its+y-Monoglutamyl+derivative+in+nonmethanogenic+archaebacteria.">Occurrence of Coenzyme F420 and Its y-Monoglutamyl derivative in nonmethanogenic archaebacteria.</a> Journal of Bacteriology. 168 (1), 444-448 (1986).</li><li>Spang, 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=The+genome+of+the+ammonia-oxidizing+Candidatus+Nitrososphaera+gargensis:+insights+into+metabolic+versatility+and+environmental+adaptations.">The genome of the ammonia-oxidizing Candidatus Nitrososphaera gargensis: insights into metabolic versatility and environmental adaptations.</a> Environmental Microbiology. 14 (12), 3122-3145 (2012).</li><li>Mand, T. D., Metcalf, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+conservation+and+hydrogenase+function+in+methanogenic+archaea,+in+particular+the+genus+Methanosarcina.">Energy conservation and hydrogenase function in methanogenic archaea, in particular the genus Methanosarcina.</a> Microbiology and Molecular Biology Reviews: MMBR. 83 (4), (2019).</li><li>Lupa, B., Hendrickson, E. L., Leigh, J. A., Whitman, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formate-dependent+H2+production+by+the+mesophilic+methanogen+Methanococcus+maripaludis.">Formate-dependent H2 production by the mesophilic methanogen Methanococcus maripaludis.</a> Applied and Environmental Microbiology. 74 (21), 6584-6590 (2008).</li><li>Kulkarni, G., Mand, T. D., Metcalf, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+conservation+via+hydrogen+cycling+in+the+methanogenic+archaeon+Methanosarcina+barkeri.">Energy conservation via hydrogen cycling in the methanogenic archaeon Methanosarcina barkeri.</a> mBio. 9 (4), (2018).</li><li>Purwantini, E., Gillis, T. P., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+F420-dependent+glucose-6-phosphate+dehydrogenase+in+Mycobacterium+and+Nocardia+species,+but+absence+from+Streptomyces+and+Corynebacterium+species+and+methanogenic+Archaea.">Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence from Streptomyces and Corynebacterium species and methanogenic Archaea.</a> FEMS Microbiology Letters. 146 (1), 129-134 (1997).</li><li>Purwantini, E., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+novel+coenzyme+F420-dependent+glucose-6-phosphate+dehydrogenase+from+Mycobacterium+smegmatis.">Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.</a> Journal of Bacteriology. 178 (10), 2861-2866 (1996).</li><li>McCormick, J. R. D., Morton, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+of+cosynthetic+factor+I+of+Streptomyces+aureofaciens+and+fragment+FO+from+coenzyme+F420+of+Methanobacterium+species.">Identity of cosynthetic factor I of Streptomyces aureofaciens and fragment FO from coenzyme F420 of Methanobacterium species.</a> Journal of the American Chemical Society. 104 (14), 4014-4015 (1982).</li><li>Coats, J. H., Li, G. P., Kuo, M. -. S. T., Yurek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+production,+and+biological+assay+of+an+unusual+flavenoid+cofactor+involved+in+lincomycin+biosynthesis.">Discovery, production, and biological assay of an unusual flavenoid cofactor involved in lincomycin biosynthesis.</a> The Journal of Antibiotics. 42 (3), 472-474 (1989).</li><li>Bown, L., Altowairish, M. S., Fyans, J. K., Bignell, D. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+the+Streptomyces+scabies+coronafacoyl+phytotoxins+involves+a+novel+biosynthetic+pathway+with+an+F420+-dependent+oxidoreductase+and+a+short-chain+dehydrogenase/reductase.">Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase.</a> Molecular Microbiology. 101 (1), 122-135 (2016).</li><li>Gurumurthy, M., 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=A+novel+F(420)+-dependent+anti-oxidant+mechanism+protects+Mycobacterium+tuberculosis+against+oxidative+stress+and+bactericidal+agents.">A novel F(420) -dependent anti-oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents.</a> Molecular microbiology. 87 (4), 744-755 (2013).</li><li>Greening, 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=Mycobacterial+F420H2-dependent+reductases+promiscuously+reduce+diverse+compounds+through+a+common+mechanism.">Mycobacterial F420H2-dependent reductases promiscuously reduce diverse compounds through a common mechanism.</a> Frontiers in Microbiology. 8, 1000 (2017).</li><li>Mathew, S., Trajkovic, M., Kumar, H., Nguyen, Q. -. T., Fraaije, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enantio-+and+regioselective+ene-reductions+using+F420H2-dependent+enzymes.">Enantio- and regioselective ene-reductions using F420H2-dependent enzymes.</a> Chemical Communications. 54 (79), 11208-11211 (2018).</li><li>Ney, B., 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=Cofactor+tail+length+modulates+catalysis+of+bacterial+F420-dependent+oxidoreductases.">Cofactor tail length modulates catalysis of bacterial F420-dependent oxidoreductases.</a> Frontiers in Microbiology. 8, 1902 (2017).</li><li>Grinter, R., 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=Cellular+and+structural+basis+of+synthesis+of+the+unique+intermediate+dehydro-F420-0+in+mycobacteria.">Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria.</a> mSystems. 5 (3), (2020).</li><li>Peck, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+concentrations+of+coenzyme+F420+analogs+during+batch+growth+of+Methanosarcina+barkeri+and+Methanosarcina+mazei.">Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei.</a> Applied and Environmental Microbiology. 55 (4), (1989).</li><li>Gorris, L. G., vander Drift, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofactor+contents+of+methanogenic+bacteria+reviewed.">Cofactor contents of methanogenic bacteria reviewed.</a> BioFactors. 4 (3-4), 139-145 (1994).</li><li>Bair, T. B., Isabelle, D. W., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+coenzyme+F(420)+in+Mycobacterium+species.">Structures of coenzyme F(420) in Mycobacterium species.</a> Archives of Microbiology. 176 (1-2), 37-43 (2001).</li><li>Portillo, M. C., Gonzalez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moonmilk+deposits+originate+from+specific+bacterial+communities+in+Altamira+Cave+(Spain).">Moonmilk deposits originate from specific bacterial communities in Altamira Cave (Spain).</a> Microbial Ecology. 61, (2011).</li><li>Wagner, A. O., 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=Medium+preparation+for+the+cultivation+of+microorganisms+under+strictly+anaerobic/anoxic+conditions.">Medium preparation for the cultivation of microorganisms under strictly anaerobic/anoxic conditions.</a> Journal of Visualized Experiments: JoVE. (150), e60155 (2019).</li><li>Lackner, N., Hintersonnleitner, A., Wagner, A. O., Illmer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogenotrophic+methanogenesis+and+autotrophic+growth+of+Methanosarcina+thermophila.">Hydrogenotrophic methanogenesis and autotrophic growth of Methanosarcina thermophila.</a> Archaea. 2018 (5), 1-7 (2018).</li><li>Wagner, A. O., Reitschuler, C., Illmer, P. <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+different+acetate:+Propionate+ratios+on+the+methanogenic+community+during+thermophilic+anaerobic+digestion+in+batch+experiments.">Effect of different acetate: Propionate ratios on the methanogenic community during thermophilic anaerobic digestion in batch experiments.</a> Biochemical Engineering Journal. 90, 154-161 (2014).</li><li>Wagner, A. O., 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=Sample+preparation,+preservation,+and+storage+for+volatile+fatty+acid+quantification+in+biogas+plants.">Sample preparation, preservation, and storage for volatile fatty acid quantification in biogas plants.</a> Engineering in Life Sciences. 17 (2), 132-139 (2017).</li><li>Bashiri, G., Rehan, A. M., Greenwood, D. R., Dickson, J. M. J., Baker, E. N. <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+cofactor+F420+production+in+Mycobacterium+smegmatis.">Metabolic engineering of cofactor F420 production in Mycobacterium smegmatis.</a> PloS one. 5 (12), 15803 (2010).</li></ol>

为了评估来自产甲烷纯培养物的辅因子F420 ，可以进行显微镜评估以可视化受累微生物的生长和活性（荧光显微镜）（图1）。对于来自自然环境的样品，由于与其他荧光微生物，有机和无机颗粒的干扰，使用显微镜检测或定量F420 受到限制。在这种情况下，如前所述，使用HPLC提取F420 和随后的荧光分析5不仅可以提供有关辅因子F4...

F420是一种广泛但经常被忽视的辅因子，在古菌和细菌的一次和二次代谢过程中作为专性双电子氢化物载体起作用1，2。F420是一种5-脱氮黄素，在结构上与黄素相似，其化学和生物学特性与NAD +或NADP +更具可比性。由于氮在异丙沙嗪环的位置5处用碳代替，它是一种强还原剂，因此表现出-340 mV1，3的低标准氧化还原电位。F420包括一个5-去氮杂黄素环和一个2-磷酸-L-乳酸接头（F420-0）。含有n+1谷氨酸单体的低聚谷氨酸尾部可以附着在分子（F420-n+1）4上。
长期以来，辅因子F420仅与古菌和放线菌有关。这在很大程度上已被推翻。最近的分析显示，F420分布在变形杆菌门，Chloroflexi和潜在的厚壁动物的各种厌氧和好氧生物中，这些生物栖息在土壤，湖泊和人类肠道等无数栖息地1，5。2019年，Braga等人6表明，Paraburkholderia rhizoxinica蛋白杆菌产生独特的F420衍生物，含有3-磷酸甘油酯而不是2-磷酸乳酸盐尾巴，这可能在各种栖息地广泛分布。在古菌域内，F420已在几个谱系中被发现，包括产甲烷7，嗜甲烷细胞8，9和硫酸盐还原目10，并且应该在Thaumarchaeota11中产生。F420最广为人知的是氢营养和甲基营养性甲烷生成中必需的氧化还原辅酶。F420（F420H2）的还原形式作为电子供体用于还原亚甲基四氢甲基蝶呤（亚甲基-H4MPT，Mer）和甲基-H4MPT12，13。它还可以用作含有细胞色素的产甲烷菌的H2非依赖性电子转运途径中的电子载体12，14。此外，F420的氧化形式在420nm处激发时具有特征性的蓝绿色荧光，这有助于在显微镜下检测产甲烷菌（图1）。由于其低氧化还原潜力，F420有助于（i）广泛谱顽固或有毒有机化合物的外源性还原，（ii）链霉菌（放线菌门）中四环素和林科斯酰胺抗生素或植物毒素的合成，以及（iii）对分枝杆菌（放线菌门）中的氧化或亚硝化应激或其他不利条件的抵抗力1，5，15，16，17，18，19，20，21，22。因此，F420依赖性氧化还原酶是有前途的生物催化剂，用于工业和制药目的以及污染环境的生物修复1，23。尽管最近有这些发现，但辅因子F420的确切作用在放线菌或其他细菌门中仍然知之甚少。
F420生物合成至少有三种途径2，6，24。首先，生物合成途径被分成5-脱氮黄素生物合成和2-磷酸乳酸盐代谢分支。F420分子的反应部分通过FO合成酶合成，使用底物酪氨酸和5-氨基-6-核氨基-2，4（1H，3H）-嘧啶二酮。结果是核黄素水平的发色团FO。在目前接受的乳酸代谢分支中，L-乳酸通过L-乳酸激酶（CofB）磷酸化为2-磷酸-L-乳酸;反过来，2-磷酸-L-乳酸通过2-磷酸-L-乳酸胍基转移酶（CofC）被鸟嘌呤基化为L-乳糖基-2-二磷酸-5'-鸟苷。在下一步中，L-乳糖基-2-二磷酸-5'-鸟苷通过2-磷酸-L-乳酸转移酶（CofD）与FO结合形成F420-02。最后，酶F420-0：ɣ-谷氨酰连接酶（CofE）将谷氨酸单体与F420-0结合，形成不同数量的最终辅因子623，25。不同的生物体在附着的谷氨酸残基数量上显示出不同的模式，与分枝杆菌相比，产甲烷菌的尾巴更短2，25，26。一般来说，产甲烷菌的尾巴长度从两到三个不等，在碎屑性产甲烷菌（Methanosarcina sp.）中最多有五个，而在分枝杆菌属中发现的尾巴长度从五到七个谷氨酸残基2，25，26，27不等。然而，最近的研究结果表明，长链F420与F420依赖性氧化还原酶结合，亲和力高于短链F420;此外，结合的长链F420增加了底物亲和力，但降低了相应酶的周转率23。
辅因子F420 的检测通常基于其荧光。因此，使用反相（RP）-HPLC27，28分离其寡聚谷氨酸衍生物。最近，Ney等人使用四丁基氢氧化铵作为带负电荷的谷氨酸尾的离子配对试剂，成功地增强了RP-HLPC上的分离5。在这里，我们提出了一种制备样品的方法，随后裂解，提取，纯化，分离和定量辅因子F420 不仅来自纯培养物，还来自不同的环境样品（即土壤和消化池污泥）。

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			<td>Autoclave</td>
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			<td>Biocompatible HPLC system equipped with gradient modul, oven and fluorescence detector</td>
			<td>Shimadzu</td>
			<td></td>
			<td>HPLC system</td>
		</tr>
		<tr>
			<td>Centrifuge and rotor for 50 mL &#8220;Falcon&#8221; tubes (11.000 rcf) and appropriate tubes</td>
			<td></td>
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			<td></td>
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			<td>HPLC Column: Gemini NX C18, 5 &#956;m, 150 x 3 mm</td>
			<td>Phenomenex</td>
			<td></td>
			<td>HPLC column</td>
		</tr>
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			<td>PTFE filter (pore size 0.22 &#181;m) to remove particulate matter prior HPLC analysis</td>
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			<td>Resin for SPE: Strata-X-AW 33 &#956;m as weak anion mixed-mode polymeric sorbent</td>
			<td>Phenomenex</td>
			<td></td>
			<td>weak anion mixed-mode polymeric sorbent</td>
		</tr>
		<tr>
			<td>Vacuum manifold for SPE and appropriate collection tubes</td>
			<td></td>
			<td></td>
			<td>SPE equipment</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction of cofactor f420 for analysis of polyglutamate tail length from methanogenic pure cultures and environmental samples

辅因子F420 在许多细菌和古菌分类群的一次和二次代谢中作为氢化物载体起着核心作用。辅因子以其在甲烷生成中的作用而闻名，它促进了热力学上困难的反应。由于不同生物体之间的聚谷氨酸尾巴长度不同，长度剖面分析可能是区分和表征不同生境中不同群体和途径的有力工具。在这里，该协议描述了通过应用固相萃取结合高效液相色谱分析来提取和优化辅因子F420 检测，而这些方法独立于培养或分子生物学方法。该方法用于从土壤、厌氧污泥和纯培养物中的微生物群落中获得辅因子F420 表达的额外信息，并通过峰值实验进行评估。因此，该研究成功地在受控的产甲烷纯培养物以及厌氧消化池污泥和土壤等环境样品中生成了不同的F420 尾长剖面，用于氢营养和碎裂性产甲烷菌。

嗜热膜肉和嗜热膜的纯培养物，两者都是嗜热的产甲烷古菌，在适当的培养基中生长，如前所述29，30。对于嗜热甲膜肉，甲醇被用作能量来源，而嗜热甲膜肉瘤菌生长在H2 / CO2上。通过显微镜评估检查生长，而通过气相色谱法测量甲烷（CH4）来检查活性，如前所述31。根据所提...

Watch this Scientific Journal Video about Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples at JoVE.com

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

针对纯培养物和环境样品中F420 尾长的液相色谱分离和分析，优化了从纯培养物中提取辅因子F420 的方法。

从产甲烷纯培养物和环境样品中提取辅因子F420 用于分析聚谷氨酸尾部长度

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is ...

辅因子F420在许多原核生物的一级和二级代谢中起着重要作用。在环境样品中测量这种分子可以更深入地了解其患病率和功能。该技术能够分析难处理样品材料（如污泥和土壤）中的辅因子。因此，材料的裂解和分析前的预纯是中心步骤。该协议包括样品制备过程中的不同步骤。在开始样品处理之前，必须很好地计划和准备这些步骤。例如，新鲜缓冲液的制备很重要。要开始样品裂解，将五克样品放入50毫升锥形管中。然后向样品中加入5毫升2X裂解缓冲液，并用蒸馏水将体积调至10毫升，达到每毫升0.5克的最终浓度。涡旋稀释的样品20秒，然后在121摄氏度下高压灭菌30分钟。对于干燥的样品，如森林土壤，在高压灭菌后用蒸馏水将最终体积提高到20毫升，并再次涡旋稀释的样品20秒，如图所示。样品冷却后，将样品裂解物以11， 000×g离心五分钟，并收集上清液。准备一个装满100毫克弱阴离子混合模式聚合物吸附剂的5毫升固相萃取或SPE色谱柱，用三毫升甲醇调节阴离子交换器。然后，用三毫升蒸馏水平衡阴离子交换器。将离心裂解液中多达9毫升的上清液加载到SPE柱上。用5毫升25毫摩尔乙酸氨和5毫升甲醇依次洗涤色谱柱中的杂质。洗涤后，用一毫升洗脱缓冲液洗脱辅因子F420。将烘箱预设为40摄氏度，并将荧光检测器设置为420纳米消光波长和470纳米发射波长。接下来，使用孔径为0.22微米的PTFE过滤器将SPE中洗脱的样品过滤到HPLC小瓶中。将50微升过滤样品注入HPLC系统，并使用手稿中描述的流动相组合通过梯度模式分离辅因子F420。分析辅因子F420的组成和浓度。通过显微镜验证了产甲烷菌纯培养物的生长。在相差显微镜中，可见产甲烷菌的聚集物，当辅因子F420被紫外光激发时，它们会发出蓝色光。分析不同体积嗜热膜炎培养物SPE后回收辅因子F420的峰面积。高压灭菌分解策略实现了最高的峰面积，使用压力-温度处理得出了最大的提取效率。对环境样品中尾长分布的分析揭示了环境样品中辅因子F420总浓度和F420尾长分布的差异。应用此过程时，请注意完全收集洗脱缓冲液的总体积或记录确切的流经体积。该技术为在土壤和污泥等更复杂的样品中探索辅因子F420铺平了道路，并能够更深入地了解该分子的生态影响。

Research

JoVE Journal

Environment

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

科研

环境科学

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

准备和测试阻抗为基础与RTgill-W1细胞饮用水进行快速评估水样进行毒性射流生物芯片

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

提取及土壤微生物磷脂脂肪酸分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...