作者感谢J. Priscu，A. Chiuchiolo和麦克默多LTER网络湖沼队在南极样品的收集和保全的协助。我们感谢Ratheon极地后勤支援服务和PHI的直升机。光显微镜在迈阿密的先进的显微镜和影像中心为中心的产生。这项工作是由国家科学基金会极地项目办公室资助0631659和1056396的支持。

1。样品采集<ol><li>选择和准备采样点有一天前采样水柱。这将使水柱分层层扰动后，由于钻井和冰孔熔化改革。识别GPS演练现场的位置。 </li><li>要访问的水柱，首先通过一个瞬间冰到了4英寸的jiffy的飞行延伸和钻头的螺旋冰钻了一个洞。为了防止冻结孔钻，尽量避免停止钻探约4-6英寸以上水柱顶部钻成液态水。 </li><li>钻孔将需要从15至60厘米，直径扩大采样前。这是通过使用一个洞熔炉（Hotsy模型），通过铜管循环加热的聚乙二醇。孔熔化通常需要长达18个小时。 </li><li>采样前的水柱，确保所有必需的设备和样品STorage船只在采样现场组装。抽样材料包括：瓶（5-10升容量）Niskin，深度校准电缆绞车，信使，每个采样深度足够的样本瓶和运输样品冷却器。 </li><li>开始采样年初的一天（5分），湖水过滤应抽样一天完成。使用连接到一个校准Niskin采样，收集所需深度（本研究为：6米，15米和18米采样深度，在冰孔测压水位测量）的水样（1-5升）手摇绞车。为了防止干扰的水柱收集之前更深的深度浅。 </li><li>店湖样品冷却器和运输领域的实验室，使用连接到ATV（全地形车）乘雪橇从样品现场。样品应处理或稳定（如：闪光灯在液态氮冷冻）在现场实验室后立即采样。 </li></ol> 2。高新区发展NT的富集文化<ol><li>为培养富集培养，集中到47毫米0.45微米孔径聚醚使用温和过滤（&lt;0.3 ATM）过滤器0.5-1大号的湖水。如果所需的自然社会的多样性，筛选到47毫米0.45微米孔径聚醚砜过滤器的第二个样品，并按照协议，根据Bielewicz 等 4真核生物进化多样性。 </li><li>过滤器转移至50毫升无菌猎鹰〜20毫升过滤湖水从相同的采样深度过滤器收集管中。从过滤器使用无菌吸管轻轻洗细胞。 </li><li>细胞悬液转移到25厘米的无菌细胞培养瓶。添加筛选湖水从每个样品深度收集至45毫升，以增加文化音量。设立多个每个采样深度复制如果需要不同的培养基富集。 </li><li>补充50X小号的文化瓶Bold的基础培养基，BG11和F / 2：以下文化媒体的1X终浓度terile解决方案。如果混合营养生物体的种植需要，设置了两个F/2-supplemented船只，​​并添加2-3瓶不育水稻的谷物。 </li><li>在低温（4℃）和低辐照（加入20μmol光子米-2 S -1），在一个温度控制的增长至少4周的孵化器在南极孵育培养瓶之前装运到美国实验室。如果需要，分离富集培养板250μL含有琼脂培养2周后的适度增长，媒体板块。 </li><li>对于运往美国，50毫升无菌猎鹰管转移富集文化。要求装运要求“不冻结”和“保持冷静”对航运的要求。虽然来自南极的运输过程中是比较快的（2个星期的商用空调）和高度可靠的，是有讲究器官损失的可能性在运输过程中的ISMS。因此，研究者可能想保持一个子样本前航运在运输过程中的损失，可以使某些物种进行评估。 </li><li>在抵达美国后，转让5毫升到45毫升的适度增长，在125毫升的无菌三角瓶媒体富集培养用无菌海绵上限。应保持站立在温度控制环境生长室（日生长商会，厂商VWR）在4°C/20号μmol光子米-2 S -1文化的丰富文化。文化应转移到新媒体每30天。 </li></ol> 3。从筛选富集的细胞裂解液提取<ol><li>过滤器5毫升到25毫米，使用温和的真空（&lt;10磅）滤纸GF / F滤波器的富集培养。该过滤器可存储数周，在-80°C间前化验Rubisco活性。 </li><li>用无菌钢剪刀削减过滤器，并为4-5节转让无菌镊子2毫升螺丝帽管件充满了直径为0.1毫米的锆/二氧化硅珠全的五分之一。 </li><li>过滤提取液加入1.25毫升（1 mM的EDTA，MgCl 2的 10毫米，50毫米bicine，5毫克/毫升BSA和0.1％的Triton X-100补充新鲜：10毫米碳酸氢钠 ，10毫米数码地面电视，3.3毫米的氨基酸，苯甲脒0.7毫米，pH值7.8）13，破坏细胞，使用Minibead搅拌器速度设置48 30年代的三倍。 1分钟冰孵化，以防止加热样品，备用beadbeating。 </li><li>在4°C离心过滤浆GF / F过滤纤维和细胞壁碎片，形成一个松散的颗粒，在3000 XG 2分钟。 </li><li>取出200μL分装在800μL冰冷的90％丙酮和浸泡。测量提取叶绿素（CHL）在分光光度计在波长664 nm和647 nm的14值。将上清转移至1.5 mL离心管中。 </li><li>离心remaini吴上清在4°为15,000 XG和2分钟，将上清转移（避免不溶性细胞膜沉淀和过滤纤维，余下的GF /）到一个干净的1.5 mL离心管中，这种可溶性的细胞裂解液，现在可能被用于在Rubisco的羧化酶活性测定。 </li><li>可存放于-80°C后，另外12.5％的甘油和闪光冷冻在液氮裂解。一个失去活性后，可能会出现多个冻结/解冻周期取决于裂解酶的敏感性。因此，新鲜与冷冻裂解活性的大规模细胞裂解提取前应测试。 </li></ol> 4。 Rubisco的羧化酶活性筛选检测<ol><li>转移125μL15 mL的螺旋盖离心管（不需要CAP）已在冰上可溶性裂解。为阴性对照样品，添加125μL，溶于裂解15毫升螺丝帽离心管，但不加衬底（步骤4.7）。运行复制所有样品和阴性对照反应。 </li><li>准备10毫升检测缓冲区新鲜（50毫米Bicine-50毫米25毫米MgCl 2的碳酸氢钠 ，氢氧化钠，pH值8.0）和冰浴。分装足够的缓冲液的所有样品和阴性对照组（100μL每采样/控制，加100μL的额外）5 mL管。通风柜下执行所有的后续步骤。 </li><li>加入40微升的NaH 14 CO 3（500微居里/毫升）每毫升缓冲液分装缓冲区和保持冰缓冲区。 </li><li>取出10μL检测含有碳-14的缓冲区，并在1毫升缓冲液稀释。经过相混合，添加100μL1:100稀释的混合物3毫升生物安全II闪烁计数闪烁小瓶鸡尾酒。反相混合。闪烁计数（贝克曼LS6500）应该超过18,000 CPM之前继续进行检测。 </li><li>当有足够多的NaH 14 CO 3的已加入缓冲液，将反应管干浴预设为3-5分钟，25℃孵育。 </li><li>加入100μL的裂解缓冲液孵育5分钟，在25°C。 </li><li>加入20μL15毫米核酮糖二磷酸，来样，使反应进行5-10分钟25°C。 </li><li>加入100μL丙酸（100％）（费舍尔）停止反应。在2000 XG离心1小时，用尽非法人14 C </li><li>转移样品的总体积闪烁小瓶含3毫升生物安全II闪烁计数鸡尾酒。确定由闪烁计数（B.维特，R. Tabita个人通信）CPM酸稳定的高端产品。 </li><li>计算Rubisco活性，叶绿素基础 14。 </li></ol> 5。代表结果我们利用富集培养分离冷适应的光合和混合营养中居于南极大号的原生生物AKE波尼。为了捕捉一个更大的生物体的多样性，我们测试三个生长介质类型：大胆的基础中等（BBM）15，F/2-Si海洋中等16，17和BG11中等18。光镜的丰富文化的视觉检查发现，光合原生生物（在大多数细胞色素叶绿素的存在表示）为主的文化和窝藏了多种采样深度取决于细胞形态的接种和文化的媒体类型（ 图1）。 我们测量了催化的酶Rubisco的羧化酶活性的最大固碳潜力的代理费率。叶绿素a丰也被监测作为光合原生动物生物量的估计，（ 图2）。尽管所有的文化修养，在相同的温度/光照制度（即4°C/20号μmol米-2秒-1），固碳潜力和光合生物量变化显着的丰富文化之间。最大Rubisco活性，观察生长在任的BBM（富集4和6）或BG11（富集13和14）生长介质中富集文化，丰富的F / 2培养基中（富集19，21，23），而文化上展出了4 - 到34倍，最大羧化酶的活动。这些差异不是由于生物量水平较低的F / 2文化，羧化酶活性，叶绿素的基础上表示。此外，Rubisco活性，叶绿素浓度 （R = -0.023，P&gt; 0.1;图2，插图）没有关联。接种湖水从6米和15米（富集4和5）的BBM文化最高的叶绿素 a水平，而所有其他文化表现出相对较低的叶绿素，无论Rubisco的酶的活性（ 图2）。 <img alt="图1" src="/files/ftp_upload/3992/3992fig1.jpg" /> 图1。代表微生物真核生物浓缩文化，而选择不同生物体的生长。文化认同是在面板的左下角。所有的图像，代表产生的光显微镜1000X倍率的油浸。 <img alt="图2" src="/files/ftp_upload/3992/3992fig2.jpg" /> 图2。潜在的固碳能力和在南极微生物真核生物富集分离湖波尼和文化上的各种文化传媒种植提取叶绿素a水平。文化的细节，请参阅表1 插图：叶绿素含量之间的相关性。皮尔逊在南极微生物真核生物的富集文化的丰富性和固碳潜力。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Description+of+Seven+Antarctic+Marine+Gymnamoebae+Including+a+New+Subspecies,+Two+New+Species+and+a+New+Genus:+Neoparamoeba+aestuarina+antarctica+n.+subsp.,+Platyamoeba+oblongata+n.+sp.,+Platyamoeba+contorta+n.+sp.+and+Vermistella+antarctica+n.+gen.+n.+sp.">A Description of Seven Antarctic Marine Gymnamoebae Including a New Subspecies, Two New Species and a New Genus: Neoparamoeba aestuarina antarctica n. subsp., Platyamoeba oblongata n. sp., Platyamoeba contorta n. sp. and Vermistella antarctica n. gen. n. sp.</a> Journal of Eukaryotic Microbiology. 54, 169-183 (2007).</li><li>Rose, J. M., Vora, N. M., Countway, P. D., Gast, R. J., Caron, 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=Effects+of+temperature+on+growth+rate+and+gross+growth+efficiency+of+an+Antarctic+bacterivorous+protist.">Effects of temperature on growth rate and gross growth efficiency of an Antarctic bacterivorous protist.</a> The ISME journal. 3, 252-260 (2009).</li></ol>

我们什么都没有透露。

最近的分子研究报告通过一系列环境3，19，20高的单细胞真核生物的多样性;然而，由于缺乏整个完整的原生生物栖息地范围的分离，这些个别物种食物链的职能作用在很大程度上是未知之数。在这项研究中，我们所描述的方法，以丰富的真核生物的参展环境，从一个相对欠永久冰雪覆盖的南极湖泊的代谢多功能的微生物物种。从不同采样深度湖波尼丰富的文化表现差的固碳率​​，生长?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> BBM协会</td><td>西格玛</td><td> B5282 </td><td></td></tr><tr><td> BG11 </td><td>西格玛</td><td> C3061 </td><td></td></tr><tr><td>的F / 2 </td><td>西格玛</td><td> G9903 </td><td></td></tr><tr><td> GF /过滤器，25毫米</td><td> Fisher Scientific则</td><td> 09-874-64 </td><td></td></tr><tr><td> GF / F滤波器，47毫米</td><td> Fisher Scientific则</td><td> 09-874-71 </td><td></td></tr><tr><td>聚醚砜过滤，0.45μm孔径，47毫米; </td><td>颇尔生命科学</td><td> 61854 </td><td></td></tr><tr><td>无菌细胞培养瓶，25厘米2 </td><td>康宁</td><td> 430639 </td><td></td></tr><tr><td> diurna升增长室</td><td>厂商VWR </td><td> 35960-076 </td><td></td></tr><tr><td>氧化锆/二氧化硅珠，0.1毫米diamter， </td><td> BioSpec产品</td><td> 11079101z </td><td></td></tr><tr><td>迷你珠打浆机</td><td> BioSpec产品</td><td> 3110BX </td><td></td></tr><tr><td>螺旋盖离心管（1.5微升） </td><td>美国科学</td><td> 1415-8700 </td><td></td></tr><tr><td>的NaH 14 CO 3的 </td><td> ViTrax </td><td> VC的194 </td><td>在400μL等份保持在-20°C </td></tr><tr><td> RuBP </td><td>西格玛</td><td> R0878-100毫克</td><td>溶解在10毫米的Tris-丙酸（pH值6.5） </td></tr></tbody></table>

establishment of microbial eukaryotic enrichment cultures from a chemically stratified antarctic lake and assessment of carbon fixation potential

波尼湖是众多永久位于麦克默多干谷，南极洲冰层覆盖的湖泊之一。常年冰盖保持化学分层的水柱，不同于其他内陆水体，主要是防止外部输入从河流的碳和营养。生物群暴露在众多的环境压力，包括全年的养分严重缺乏，气温低，极端的阴凉处，矿化，24小时黑暗，在冬季1。这些极端的环境条件限制在波尼湖生物群几乎完全微生物2。 单细胞微生物的真核生物（称为“原生生物”）是在全球生物地球化学循环的重要球员，在干热河谷湖泊的碳循环中发挥重要的生态作用，占小学和三级水生食物网中的角色。在干热河谷水产食品网站，修正我的原生生物organotrophic生物体的有机碳4，2 norganic碳（自养）的主要生产者。 phagotrophic或能异养细菌和较小的原生生物摄入原生生物作为食物网中的顶级掠食者。最后，未知的原生动物人口的比例是能够结合的混合营养代谢6，7。在原生生物mixotrophy涉及的能力结合猎物微生物phagotrophic摄入的光合能力。这种形式的mixotrophy不同于细菌混合营养代谢，一般涉及吸收溶解的碳分子。目前有极少数原生动物分离永久冰覆盖的极地湖泊，在这种极端环境中的原生生物多样性和生态的研究已经有限，8，4，9，10，5。的原生生物代谢的多功能性，将有助于更好地理解简单的干热河谷湖的食物网中的发展模式，为rOLE在全球碳循环中的原生生物。 我们采用隔离波尼湖潜在的光合和混合营养的原生生物富集培养方法。水柱采样深度被选为基于初级生产最大值和原生动物进化多样性4，第11的位置，以及影响原生动物的营养模式的主要非生物因素的变化：浅层取样深度为主要营养成分的限制，而更深层次的采样深度是有限的可用光。此外，湖水样品，辅以多种类型的生长介质，促进多种光合生物体的生长。 Rubisco活化酶催化的速率限制在CBB）卡尔文森Bassham（周期，主要途径，其中自养有机体修复无机碳和有机碳含量较高的营养水平，在水生和陆生食物链12步。在这项研究中，Wé应用放射性同位素法修改过滤样品13监察固碳潜力，并在湖的波尼富集文化的代谢多功能的代理最大的羧化酶活性。

Watch this Scientific Journal Video about Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential at JoVE.com

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

微生物的真核生物都在永久性冰雪覆盖的南极湖泊源的光合衍生的碳和顶级掠食性物种。本报告介绍了富集培养的方法分离代谢多功能微生物真核生物，从南极湖，湖波尼，使用核酮糖1,5 - 二磷酸羧化酶加氧酶（Rubisco活化酶）活性放射性同位素检测和评估无机碳固定潜力。

建立真核微生物的富集培养从化学分层南极湖和固碳潜力评估

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

establishment-microbial-eukaryotic-enrichment-cultures-from

Research

JoVE Journal

Biology

11.3K 次观看.  Miami University. 微生物的真核生物都在永久性冰雪覆盖的南极湖泊源的光合衍生的碳和顶级掠食性物种。本报告介绍了富集培养的方法分离代谢多功能微生物真核生物，从南极湖，湖波尼，使用核酮糖1,5 - 二磷酸羧化酶加氧酶（Rubisco活化酶）活性放射性同位素检测和评估无机碳固定潜力。

视频: Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

NK细胞与干细胞技术的RosetteSep套件的人体血液的富集

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

科研

生物学

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

使用比色皿型快速重复率荧光计测量 Colacium sp.附着阶段的光生理学

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

从蜱 离体 唾液腺培养物和细胞外囊泡中分离microRNA

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury

To begin, prepare two milliliters of 4%formalin or any other appropriate fixative. Also keep a petri dish, forceps, and surgical scissors ready before starting the procedure. Place the properly euthanized cryoinjured fish in a petri dish containing deionized water.Using scissors, perform a cut through the body, anterior and posterior to the caudal peduncle. Let the tissue bleed out in the deionized water inside the petri dish. Using forceps, collect and transfer the caudal peduncle into the prepared fixative solution in a microcentrifuge tube.Carefully invert the tube several times. Before mounting, wash the fixed tissue in PBS for 10 minutes on a rocker. Then transfer it into a two-milliliter microcentrifuge tube containing a solution of 30%sucrose in deionized water, pre-cooled to four degrees Celsius and gently invert the tube several times.Leave the tube upright at 4 degrees Celsius for at least 24 hours. Fill an embedding mold with a five-millimeter layer of OCT mounting medium. Using forceps, place the caudal peduncle at the bottom of the mold.Adjust its orientation for either transversal or coronal sections. Let the medium freeze in a box of dry ice. As soon as the tissue is stabilized in the desired position, fill up the rest of the mold before the OCT completely freezes over.Store the mold at 80 degrees Celsius for at least one hour before sectioning the tissue using a cryostat. The extent of cryoinjury at different days post-cryoinjury, or DPCI, was analyzed in the caudal peduncle sections using trichrome staining composed of Aniline Blue, Acid Fuschin, and Orange-G. The damaged areas were determined by the absence of orange staining.At 4 and 7 dpci, the transverse section displayed extensively degenerated skeletal muscle in the cryoinjured flank of the body. Immunofluorescence analysis showed that, at 4 DPCI, the injured side of the caudal peduncle contained abundant DAPI-positive cells but little to no F-actin and tropomyosin 1, indicating degenerated muscles. At 7 dpci, tropomyosin 1 and F-actin could be detected in the wound, close to the vertical body midline, indicating the start of new myofiber formation.At 30 dpci, both sides of the body displayed a similar distribution of F-actin staining, indicating efficient skeletal muscle restoration.