Name: an optimized enrichment technique for the isolation of arthrobacter bacteriophage species from soil sample isolates
Uploaded: 2015-04-09T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates at JoVE.com

资助这项协议的发展是由东南宾夕法尼亚财团高等教育和卡布里尼学院科学系提供。额外的资金和支持来自阿卡迪亚大学和Immaculata大学。我们特别感谢莫文蔚Snetselaar博士在圣约瑟夫大学好心参加我们的孤立的噬菌体电子显微图像。额外的支持是由霍华德·休斯医学研究所科学教育联盟噬菌体猎人推进基因组学和进化科学（SEA-噬菌体）计划提供的。

1.制备Arthobacter细胞对噬菌体分离<ul><li>培养节杆菌。KY3901菌落划线上的Luria Bertaini（LB）琼脂平板上培养，在30℃下进行2-3天。选择一个菌落并使用无菌环到它在225转添加250ml的LB肉汤中在带挡板的培养瓶中，并培育在振荡培养箱中于30℃。 </li><li>允许约24小时的生长，得到迟指数/早期静止期细胞噬菌体感染实验。监测细菌生长的状态密切以防止细胞进入到中间后期静止生长状态。 注意:细胞生长应0.5-.0.7对于下面描述的噬菌体的分离和纯化步骤/步骤之间包括一个光密度OD 600。 </li></ul>从土壤样品2.收集噬菌体<ol><li>收集200-400克土壤所需的位置。注:由于细菌在ArthrobaCTER属是常见的土壤细菌，他们和他们感染的噬菌体中可以找到并普遍存在于大多数类型的土壤。 </li><li>添加到土壤至少400毫升噬菌体缓冲液（PB）[68 mM氯化钠，的10mM 硫酸镁 ，10毫摩尔Tris-CL（pH为7.5）]，并混合在一个足够大的烧瓶，使至少200毫升的PB是在上清液中，并能够被提取。 </li><li>轻轻搅拌或涡旋直至土壤悬浮混合，并且允许在土壤沉积物沉降，一般为30分钟。 </li><li>通过重力过滤通过"土壤提取物"，通过过滤纸，除去泥沙土屑。 </li><li>添加LB粉末并混合，使2％的溶液（4克的LB粉末在200毫升土壤提取物）。 </li><li>通过真空过滤通过0.22微米过滤的溶液，得到的无菌滤液。如果可能的话，请继续，立即步骤2.7。可行的噬菌体颗粒，如果需要的话，店内过滤样品在4℃长达一个星期前进行，然而，数会降低。 </li><li>多个等分试样（每份50ml）的过滤灭菌的LB /土壤提取物的混合物的成单个250毫升灭菌摇使用无菌技术挡板的培养瓶中。因为不同的节杆菌噬菌体不同的CaCl 2的条件下生长最佳添加无菌的2.0M的CaCl 2溶液至最终所需的浓度（0-50毫米）。 注意:我们发现，多数确定我们的节杆菌噬菌体的是为1mM-2.5mM的氯化钙2的范围内。然而，一些孤立的噬菌体的增长我们专门在0毫米氯化钙2。或者在非常高的氯化钙浓度。立即进行步骤3.1。 </li></ol> 3.噬菌体隔离<ol><li>加12.5毫升晚指数/早期稳定期细菌培养物到每个烧瓶。对于OD 600从0.5-0.7，使用1毫升细胞。对的OD 600高于0.7的OD使用将2.5ml细胞。 注:指数生长期细胞通常产生更多的噬菌体和更高的滴度比细胞静止期增长。 </li><li>在振荡培养箱摇瓶中以250rpm在30℃下大约24小时。 </li><li>经过24小时的潜伏期从摇床删除富集瓶和稀释的浓缩样品十倍噬菌体缓冲。 </li><li>建立培养管0.5下旬指数/早期固定相细菌培养毫升，加入不同量的富集培养（5微升至500微升10 -1稀释PB），以文化管。 注意:最好是测试各种浓度的，因为每个土样富集后会产​​生不同的噬菌体滴度。 </li><li>加入氯化钙至培养管，使最终浓度等于存在于原始富集烧瓶浓度。使用一定范围的不同的CaCl 2浓度的选择用于许多噬菌体瓦特第i个不同的氯化钙的依赖。 注:大多数噬菌体将在氯化钙范围1-2.5毫米之内孤立的，而是有噬菌体中分离最优化的任何地方从0-50毫米氯化钙2浓度。因为这一点，最好是检查范围的CaCl 2浓度最大化的独特分离节杆菌的噬菌体的数目。 </li><li>混合4.5毫升的LB顶层琼脂中培养管，将混合物倾到的LB琼脂板上。轻轻摇动整个板均匀地分布，并允许上层琼脂凝固约15分钟。 注:顶部琼脂可以在较大的批量（250毫升）和附加的顶层琼脂制备可以存储在RT并再用于随后的实验中，至少有两个星期。虽然有用来使顶层琼脂不同的公式，我们使上层琼脂使用7克/ L琼脂的LB中与混合在60℃的水浴代替顶层琼脂的实验过程中保持液态。 </li><li>反转吨他板和孵化在所需温度（温度为30°C）O / N为48小时。改变这些条件进行优化为存在的噬菌体。最孤立的噬菌体来自一个24小时的潜伏期后，培养在30℃1-2.5毫米氯化钙2浓度板。 </li></ol> 4.净化噬菌体<ol><li>经过24小时培养检查平板上斑块的形成。继续温育长达72小时，如果没有斑块是显而易见的，或以确定是否附加的噬菌斑出现。如果需要的话，店里板的假定斑块长达一个星期在4℃之前，诉讼的。 注:这是不难发现各种不同的斑块形态的一个板块。孵化后3天发现斑块是可能的，但很少见。 </li><li>净化使用条纹斑点法明确孤立斑块预期的噬菌体。触摸牌匾用无菌木棍。连胜斑块通过火焰运行木棍，allowin克棒简单冷却，然后擦在琼脂平板在柔和的运动的棒， 如图1。使用清洁棒对各遍重复此。 </li><li>另外，使用传统的牌匾滴度检测，以分离单个噬菌斑。噬菌斑测定法滴度确实需要使用多种试剂，如LB琼脂和LB顶层琼脂和材料如培养皿噬菌体斑块隔离。我们已经非常成功使用条纹菌斑的方法，但它在技术上是容易分离使用噬菌斑的滴度测定个体斑块。 </li><li>一旦在LB琼脂平板上划线接种是在至少三次，马克与推定的噬菌体颗粒的最低浓度的区域，并轻轻申请0.5毫升节杆菌和4.5毫升的LB熔融顶层琼脂（含有氯化钙的相同浓度的父板），并允许它均匀地分布在整个板。 </li><li>反转，并培育板O / N。重复连胜牌匾技术至少三次迭代，以确保一个纯噬菌体物种是分离的。商店板在4℃下进行长达一周需要条纹之间不显著损失噬菌体生存能力。 </li><li>一旦需要的斑块已经发展壮大，孤立的最终连胜板，触摸一体以及孤立的牌匾用枪头，重新悬浮于100微升PB。串联通过使20微升样品放入180微升新鲜稀释的PB这10 0的溶液出到10 -8稀释。 </li><li>在培养管中进行5加10微升每种稀释液以0.5 节杆菌生长毫升至室温10分钟，板通过混合感染的细菌具有4.5的含氯化钙的相同浓度用于分离噬菌体熔融的LB上层琼脂毫升从原来的土样。保存发在4°C，直到第二天的所有稀释。 </li><li>确定需要做出采用传统的牌匾滴度为web模式的噬菌体连续稀释说了。菌斑的滴度测定的目的是确定需要获得一个web图案板的噬菌体效价。识别网页模板的大多是缺乏细菌，但包含尚未通过裂解噬菌体细菌的残余。加入5 ml后期指数/早期固定节杆菌培养到无菌的50ml培养瓶中。 <ol><li>添加100微升的稀释液（即给一个web图案的节杆菌培养物）中培养瓶，并允许噬菌体感染的细菌细胞进行5至10分钟，在RT。混合使用作为用于最初鉴定噬菌体和板5毫升该混合物到10新鲜LB平板含有氯化钙的相同浓度的熔融的LB琼脂顶部。允许固化并孵育倒置平板，于30℃。 </li></ol></li><li>第二天，洪水幅图案板用5ml PB和商店O / N的，在4℃或4小时，在室温。 </li><li>通过0.22微米的注射器过滤器过滤该噬菌体裂解物并存储在4℃。使用此最终噬菌体股票为另外的实验，如噬菌体颗粒的电子显微镜图像，噬菌体基因组DNA分离为使用标准程序DNA的限制酶分析和基因组测序。对于长期储存的噬菌体股票，噬菌体裂解液等量的小药瓶股票归档于-80℃加入到无菌甘油。 注:对于那些没有经历过这些噬菌体技术，一个巨大的资源是在线访问www.phagesdb.org网站。 </li></ol>

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尽管许多以前曾试图孤立能够感染主机节杆菌噬菌体的，我们有一点成功使用标准的浓缩过程。细菌富集开发并适于通过面包车Twest和Kropinski 10从环境样品富集的噬菌体的广义方法仍是依据对大多数富集过程。从以往的研究证据表明，直接电镀的方法都对节杆菌菌株的检测斑块尽管有隔离5的成功率极低。与直接镀法，噬菌体是从土壤样品中提取噬菌体缓冲和过滤，以...

节杆菌种在土壤环境中无处不在提供能够从这种寄主细菌被噬菌体分离出一个庞大的数量和多样性。在Acintobacteriaceae家族的细菌成员是最显着的降低顽固的化合物，如莠去津和其他各种杀虫剂和除草剂1,2,3他们的代谢途径。虽然大多数研究已经完成使用的节杆菌菌株环境，本属临床分离株血，尿，眼睛，和其他许多人来源的所有显示系统发育不均匀4找到。 虽然有相当广泛的对节杆菌的细菌研究机构，只有少数研究能够感染这种多样化属成员的噬菌体报告。但有趣的是，在此前节杆菌所做的工作噬菌体几个关键的不同的主题接触，如土壤类型&lt;EM&gt;节杆菌物种5，工业用途以减少有害的泡沫在活性污泥处理厂6和工作突出位点特异性重组和整合基因7的目的。 各种富集技术协议已被利用来产生纯的噬菌体分离物在节杆菌物种。早期的程序包括土壤添加了有毒物质尼古丁一样的盐每年超过8引起的时期孵化到能够只感染A的噬菌体球形节杆菌 。研究使用的土壤渗出完成与不稳定的有机物经出现斑块检测技术，生产检测噬菌体，省略了漫长的潜伏期8。但有趣的是，一个类似的技术直接电镀用在过去的几个噬菌体引起同时还具有一个显着低成功率的调查5，理由是低成功率过去的研究<s达&gt; 8。 总体而言，在过去所用的分离技术是值得注意的具有尽管节杆菌属的代表最常见的有氧土壤在实践中很少功效隔离性质的4,9，，范Twest和Kropinski 10本富集方法用于从水和土壤中分离的噬菌体改编自用于充实环境的细菌菌株，但这些富集技术的早期技术被证明效率低下的隔离节杆菌噬菌体。这里所描述的方法的目的是要表明"概念验证"，早期的富集方法可以适于一致地和有效地隔离节杆菌噬菌体，克服了与来自该细菌属中分离的噬菌体相关的先前的技 ​​术挑战。

<table><tbody><tr><td>LB Broth powder</td><td>Fisher</td><td>BP9722-2</td><td>It&#039;s best to order these in bulk.</td></tr><tr><td>Granulated Agar</td><td>Fisher</td><td>BP1423-2</td><td>It&#039;s best to order these in bulk.</td></tr><tr><td>0.22 um syringe filters</td><td>Fisher</td><td>09-719A</td><td></td></tr><tr><td>0.22 um buchner filters</td><td>Fisher</td><td>430320</td><td>More than 50 ml of liquid can be obtained by carefully swapping the receiving tube.</td></tr><tr><td>Eppendorf Tubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>5 ml pipets individual</td><td>Fisher</td><td>13-678-11D</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Fisher</td><td>76002844</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Fisher</td><td>76002845</td><td></td></tr><tr><td>10 ml pipets individual</td><td>Fisher</td><td>13-676-10J</td><td></td></tr><tr><td>25 ml pipets individual</td><td>Fisher</td><td>13-676-10K</td><td></td></tr><tr><td>Whatman qualitative filter paper</td><td>Fisher</td><td>1001-824</td><td></td></tr></tbody></table>

an optimized enrichment technique for the isolation of arthrobacter bacteriophage species from soil sample isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

为了证明对节杆菌噬菌体改进富集技术的再现，2014年这些独特的节杆菌噬菌体从采集的土壤样品22使用这种浓缩获得30土壤样品的春季和夏季期间，30个不同的土壤样品用于在不同的时间和地点流程。标准的富集过程产生独特的噬菌体3相同的土壤样品。所述富集的样品可具有非常高的噬菌体滴度需要初始稀释以分离斑块或相对低的噬菌体滴度要求富集的节杆菌的较大体积成功?...

Watch this Scientific Journal Video about An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates at JoVE.com

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

一种优化富集技术的隔离<em&gt;节杆菌</em&gt;噬菌体种从土壤样品隔离

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

an-optimized-enrichment-technique-for-isolation-arthrobacter

Research

JoVE Journal

Environment

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

19.4K 次观看.  University of the Sciences. We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

视频: An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

科研

环境科学

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

免疫与感染

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

从环境中自然产生的趋磁细菌的采集，分离和富集

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

生物学

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

噬菌体介导的莱姆病螺旋体伯氏疏螺旋体的遗传操作

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

从土壤中分离可培养酵母和霉菌以研究真菌种群结构

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

通过富集低 MW DNA 评估环境 AMR 的高效技术

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

作者聚焦：通过将基于培养的技术和基因组学相结合来了解和检测环境抗菌素耐药性

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估 

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

一种优化富集技术的隔离

摘要

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此视频中的章节

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

可扩展的高通量选择从噬菌体展示合成抗体库

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

从环境中自然产生的趋磁细菌的采集，分离和富集

噬菌体介导的莱姆病螺旋体伯氏疏螺旋体的遗传操作

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

从土壤中分离可培养酵母和霉菌以研究真菌种群结构

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估