Name: biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans
Uploaded: 2015-03-19T16:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

这项工作是由美国国家科学基金会（奖＃0920274）和南卡罗来纳州实验站项目SC-1700340奖励支持。克莱姆森大学实验站本文isTechnical贡献号6283。作者感谢卢卡斯Kozubowski博士，他在这最后的协议和谢丽尔英格拉姆 - 史密斯博士，凯蒂格伦，和Grace Kisirkoi其关键手稿阅读的发展有益的建议。

注:本协议的总体方案是在图1列出。 1. C.新型隐球菌准备<ol><li>对于每一个转化反应，增长C.一个2-3毫升的O / N培养隐球菌在YPD培养基中在30℃下250rpm摇动。 </li><li>离心的O / N培养5分钟，在900×g离心10℃并弃去上清液。 </li><li>重悬在300微升酵母蛋白胨葡萄糖（YPD）培养基的每个细胞沉淀。 </li><li>使用玻璃珠，轻轻地将300微升洗涤细胞悬浮液在含1M山梨醇的YPD琼脂上。 </li><li>允许板，以在常温下干燥3-4小时。 </li></ol> 2.金微载体的制备<ol><li>悬浮0.25克0.6微米的金珠1毫升DDH 2 O的中，离心900 XG 1分钟沉淀珠子，去除上清。 </li><li>重悬金珠在1ml的100％乙醇。&lt;/ li&gt; <li>分发珠粒成4管，250微升每种，然后添加750微升的100％乙醇。 </li><li>商店金珠等分在4℃。 </li></ol> 3. DNA准备<ol><li>通过浸没其中在100％乙醇使用镊子制备橙大载体基因枪光盘。地方光盘插入含有燥石膏干大培养皿（确保燥石膏不接触光盘）。 </li><li>一旦干燥，按大载体盘入银盘支架（先前擦平用100％乙醇）。 </li><li>涡旋金珠（其制备如上述步骤2）和等分12微升到1.5 ml离心管，每1转化管。 </li><li>添加到每个管中顺序:2的DNA的微克（优选2微升的1微克/微升的DNA），10微升的2.5M的CaCl 2，和2μl的1M亚精胺游离碱。 </li><li>设置一个阴性对照如在步骤3.4，但没有的DNA。 </li><li>涡流每个管和孵化在室温下5分钟。轻轻一抖各管偶尔此孵化过程中重悬落户珠。 </li><li>旋管在225×g离心30秒以沉淀DNA包被的金颗粒。小心取出上清液（吹打或抽吸），并丢弃。 </li><li>完全在600微升的100％乙醇重悬珠子通过缓慢上下抽吸。 </li><li>旋管在225×g离心30秒以沉淀珠无填料。小心地取出并弃去上清液。 </li><li>通过慢慢地上下吹打重悬DNA包被的金颗粒在8微升的100％乙醇。 </li><li>吸管将DNA包覆的金颗粒上的1厘米直径的所述生物射弹盘的中心并且允许干燥。 注:干燥金圆上的生物射弹盘的中心可见指示金珠的足够的浓度存在。 注:装载有DNA包被金颗粒的大载体盘现在准备用于与基因枪使用。 </li></ol> 4.Ø操作摄像机基因枪<ol><li>开启真空泵上。 </li><li>转动旋钮，直到大约2200磅的压力打开氦气达到压力表。 </li><li>通过翻转的红色开关左侧打开基因枪。 </li><li>可以肯定的流速为真空和通气被调整，以便在真空将达到在15秒内28英寸汞柱。 </li><li>可以肯定的破裂盘和大载体之间的距离大约是3/8英寸。 </li><li>通过擦拭用乙醇清洁整个室。 </li><li>淹没在100％乙醇的破裂盘。允许无菌表面干上（ 例如 ，培养皿）。 </li><li>使用扭矩扳手松开爆破片固定器。将干净的爆破片到支架。爆破片支架螺丝放回原位，并一度将其向右拧紧力矩扳手。 注:安全盘会在每次拍摄所取代。 </li><li>淹没日Ë纱网在100％的乙醇。允许无菌表面干上（ 例如 ，培养皿）。 </li><li>一旦干燥，放置一个洗涤目筛上的白色塑料安装板。下来将大载体盘持有者DNA侧进盘室。拧上银帽，然后将安装板中的最高槽。 注:该网筛将分别拍摄后进行更换。 </li><li>放置含1M山梨糖醇在底板的YPD琼脂平板上。 </li><li>关上房间的门和锁到位。 </li><li>按下并按住中间的红色开关高达从事真空，并允许真空达到28英寸汞柱。一旦合适的真空度达到，将此开关向下的位置。准备就绪后，按住上的火权的红色开关。当爆破片弹出，立即释放火按钮，按下中间的红色开关至中间位置，以发泄室0磅。 </li><li>清理掉破裂盘碎片和关闭基因枪。然后关闭heliu米气体通过转动旋钮顺时针，最后，关闭真空泵关闭。 </li></ol> 5.电镀转化细胞<ol><li>允许转化板坐于RT搅拌4小时，以使细胞恢复。 </li><li>吸移管700微升的YPD的上板。用细胞刮刀轻轻刮去细胞脱落琼脂和吸管液体进入无菌1.5毫升microfuge管。重复此步骤，以确保所有的细胞已被追回从板块。 </li><li>沉淀细胞在225×g离心30秒。取出并弃去上清液。 </li><li>悬浮在500微升YPD的沉淀。 </li><li>吸移管将100μl细胞悬浮液到YPD +抗生素板的中心和散布用玻璃珠。 </li><li>离开倒板在RT 3-4天。作为殖民地的出现，补丁到新的YPD +抗生素板块。 </li></ol> 6.基因组DNA提取的PCR 注:这是使用试剂的修改版本从DNA纯化试剂盒S（见表材料）。 <ol><li>成长5毫升培养每个C.的隐球菌转化体在YPD液体在30℃下250rpm摇动O / N。 </li><li>颗粒3毫升细胞在900 XG，重悬在600微升的核裂解液。 </li><li>添加该悬浮液到一个新的1.5 ml离心管用200μl0.5mm的酸洗涤的玻璃珠。 </li><li>在小型珠磨器在环境温度下，凉爽管在冰上匀化45秒，并重复。 </li><li>允许样品定居在冰上2分钟，转移上清至新的1.5毫升管。加入200微升蛋白沉淀溶液到每个管中，（100微升每600微升上清液中回收的）和旋涡剧烈持续20秒。 </li><li>允许样品定居在冰上5分钟，并离心在11000×g离心3分钟。 </li><li>上清转移到一个干净的1.5毫升管含有300微升RT异丙醇。颠倒轻轻拌匀。 </li><li>离心机样品在11000 XG为2分钟，小心取出上清液，漏管到纸巾。 </li><li>添加300微升的RT的70％乙醇到每个管中，并轻轻颠倒以洗涤沉淀。 </li><li>在11000×g离心2分钟，并小心地离心样品除去所有的乙醇。 </li><li>排空管子上清洁纸巾，并允许沉淀在空气中干燥10-15分钟。 </li><li>加入50微升的DNA补液溶液和1.5微升RNA酶溶液，以每个颗粒和旋涡。 </li><li>离心样品5秒到从盖除去所有的液体。 </li><li>孵育的样品在37℃下进行15分钟。 </li><li>通过温育的样品在65℃下1小时再水合的DNA。 </li><li>通过测量260nm处的吸光度分光光度计定量的DNA（一个A 260读数为1.0，相当于约50微克/毫升的双链DNA），并使用高达200纳克在每个PCR反应。 </li></ol> 7. RNA分离的逆转录PCR。 <ol><li>使用RNA纯化试剂盒（见表材料），按照制造商的说明，从使用minibeadbeater酵母细胞中分离RNA。 </li><li>通过测量260nm处的吸光度定量RNA的浓度（的A 260读数为1.0，相当于〜40微克/毫升的单链RNA）。 </li><li>采用RT-PCR试剂盒（见表材料），按照制造商的说明设置了大约1微克RNA​​的RT-PCR反应。在这项研究中获得的结果，使用表1中所列的引物。 </li></ol>

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We have nothing to disclose.

Utilizing this protocol, biolistic transformation can be accomplished in which linear DNA is integrated into a desired locus in the Cryptococcus neoformans genome by homologous recombination. Certain steps in the protocol can have a dramatic effect on the effectiveness/efficiency of the transformation. For a successful transformation, it is imperative that the DNA utilized in the shoot has a concentration of at least 1 &#181;g. However, the volume of DNA added to the gold beads can be increased in the chance the...

Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis resulting in more than 625,000 deaths per year worldwide 1. Acetate has been shown to be a major fermentation product during cryptococcal infection 2,3,4, and genes encoding enzymes from three putative acetate-producing pathways have been shown to be upregulated during infection 5. This suggests that acetate production and transport may be a necessary and required part of the pathogenic process; however, the significance of this is not yet understood. One possible pathway for acetate production is the xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack), a pathway previously thought to be present only in bacteria but recently identified in both euascomycete as well as basidiomycete fungi, including&#160;C. neoformans&#160;6.
To determine the localization of these enzymes of this pathway in the cell, a construct carrying a neomycin resistance gene downstream of an ACK gene fusion to the fluorescent tag mCherry (ACK:mCherry:Neo) will be introduced into C. neoformans using the well-established method of biolistic transformation 7,8. Although electroporation is an efficient method for transformation of plasmids that will be maintained as episomes into Cryptococcus 9, it is not useful in creating stable homologous transformants 8. Only&#160;biolistic&#160;delivery using a gene gun&#160;provides&#160;an effective means to transform&#160;linear&#160;DNAs&#160;that will be integrated into the genome by homologous recombination. For example, Edman et al. showed that of the transformants resulting from electroporation of a plasmid-borne URA5 selectable marker into C. neoformansura5 mutants, just 0.001 to 0.1% of transformants were stable 9. Chang et al. achieved just a 0.25% stable transformation efficiency using electroporation to reconstitute capsule production in an acapsular mutant 10. Unlike electroporation, biolistic transformation has been shown to result in stable transformation efficiency of 2-50% depending on the gene that is being altered 7,8,11. 
This visual experiment will provide a step-by-step demonstration of biolistic transformation of the linear ACK:mCherry:Neo DNA construct into C. neoformans, and will describe how to confirm its proper integration via homologous recombination into the&#160;ack&#160;locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory 8.

<table><tbody><tr><td>0.6 &amp;mu;m gold beads</td><td>Bio-Rad</td><td>165-2262</td><td>http://www.bio-rad.com</td></tr><tr><td>Spermadine-free base</td><td>Sigma- Aldrich</td><td>S0266</td><td>https://www.sigmaaldrich.com</td></tr><tr><td>G418 - Sulfate (Neomycin)</td><td>Gold Biotechnology</td><td>G-418-10</td><td>www.goldbio.com</td></tr><tr><td>Hygromycin</td><td>Gold Biotechnology</td><td>H-270-1</td><td>www.goldbio.com</td></tr><tr><td>1350 psi Rupture Discs</td><td>Bio-Rad</td><td>165-2330</td><td>http://www.bio-rad.com</td></tr><tr><td>Stopping Screens</td><td>Bio-Rad</td><td>165-2336</td><td>http://www.bio-rad.com</td></tr><tr><td>Macrocarriers discs</td><td>Bio-Rad</td><td>165-2335</td><td>http://www.bio-rad.com</td></tr><tr><td>YPD Broth</td><td>Becton Dickinson &amp;amp; Co.</td><td>242820</td><td>www.bd.com</td></tr><tr><td>Agar</td><td>Becton Dickinson &amp;amp; Co.</td><td>214530</td><td>www.bd.com</td></tr><tr><td>Sorbitol</td><td>Fisher Scientific</td><td>BP439</td><td>http://www.fishersci.com</td></tr><tr><td>PDS-1000/He System</td><td>Bio-Rad</td><td>165-2257</td><td>http://www.bio-rad.com</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio</td><td>http://www.zeiss.com/microscopy</td></tr><tr><td>KOD One Step PCR Kit</td><td>EMD Millipore</td><td>71086-4</td><td>http://www.emdmillipore.com</td></tr><tr><td>One Step RT-PCR Kit</td><td>Qiagen</td><td>210212</td><td>www.qiagen.com</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>www.promega.com</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>www.qiagen.com</td></tr><tr><td>Mini Beadbeater - 1</td><td>BioSpecs</td><td>3110BX</td><td>http://www.biospec.com</td></tr><tr><td>Microfuge 18 Centrifuge</td><td>Beckman Coulter</td><td>F241.5P</td><td>www.beckmancoulter.com</td></tr><tr><td>Microplate Spectrophotometer</td><td>BioTek</td><td>EPOCH</td><td>www.biotek.com</td></tr></tbody></table>

biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans

所述担子菌新生隐球菌 ，中枢神经系统的一种侵入性致病菌，是真菌性脑膜炎全世界导致全世界每年超过625000例死亡的最常见的原因。虽然电穿孔已经被开发用于质粒在隐球菌的转化，生物射弹仅递送提供了转换，该转换可以集成到通过同源重组的基因组线性DNA的有效手段。 乙酸已被证明是隐球菌感染期间的一个主要的发酵产物，但这种意义尚不清楚。一种细菌途径中的酶木酮糖-5-磷酸/果糖-6-磷酸磷酸（XFP）和乙酸激酶（ACK）所组成，是用于生产乙酸在C 3潜在途径1 新型隐球菌 。在这里，我们展示了一个结构的基因枪转化，已确认编码基因融合到荧光标记mCherry，为C.新型隐球菌 。然后，我们确认整合到ACK -mCherry融合到所述ACK轨迹。

C.的成功基因枪转化隐球菌可以按照此协议方案（ 图1）而获得。用生物射弹转化，的涂覆金珠成功的射门由金环上的板可见指示后该DNA是射门（ 图2A）。在室温下电镀回收的细胞从所述的YPD + 1M山梨醇板到选择性培养基后，当左菌落出现在4至5天。转化2微克的DNA应导致20至30的菌落（ 图2B）。当菌落出现，应再划线在选择性培养基上进行单个菌?...

Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

生物射弹转化是用于产生的DNA的稳定整合入条件致病菌新生隐球菌通过同源重组的基因组中的方法。我们将展示一个结构，它具有融合到荧光标记mCherry到新生隐球菌基因编码醋酸激酶的基因枪转化。

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

biolistic-transformation-fluorescent-tagged-gene-into-opportunistic

Research

JoVE Journal

Immunology and Infection

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

9.9K 次观看.  Clemson University. 生物射弹转化是用于产生的DNA的稳定整合入条件致病菌新生隐球菌通过同源重组的基因组中的方法。我们将展示一个结构，它具有融合到荧光标记mCherry到新生隐球菌基因编码醋酸激酶的基因枪转化。 

视频: Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

在荧光蛋白融合的一代<em&gt;念珠菌</em&gt;物种

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

科研

遗传学

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

发育生物学

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

生物学

利用根癌农杆菌在小球藻中的稳定基因整合

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

作者聚焦：使用根癌农杆菌优化寻常小球藻的转化方案 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

生物工程

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

荧光标记基因的基因枪转化成机会性真菌病原

摘要

探索更多视频

此视频中的章节

在荧光蛋白融合的一代

基于荧光的感染组织细菌基因调控方法

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

基因改造的Picoeukaryote

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

荧光标记基因的基因枪转化成机会性真菌病原

摘要

探索更多视频

此视频中的章节

在荧光蛋白融合的一代

基于荧光的感染组织细菌基因调控方法

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

基因改造的Picoeukaryote

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

根癌农杆菌介导的绿色微藻、寻常小球藻的基因工程

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

用于分析 念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备

丝状蓝藻腔隙的自然转化、蛋白表达和低温保存

用于分析念珠菌 Cdr1-mGFPHis分析的小尺度等离子膜制备