作者感谢由德国研究基金会（DFG）通过合作研究中心SFB的578"从基因到产品"在德国不伦瑞克工业大学提供的财政支持。

1。栽培反应堆的安装和启动共4生物反应器栽培进行。 <ol><li> 答:种植3大号搅拌式生物反应器使用的2.2升工作体积尼日尔 SKAn1015。 </li><li>倒入到反应堆72.6克葡萄糖水合物，填补了1.9升去离子水。 </li><li>安装反应堆设备，如3挡板，两个六叶圆盘涡轮叶轮，pH电极，进气口与空气滤清器，冷却的手指，排气空气冷却器与空气过滤器，空气过滤器的无菌采样浸入管为培养基，接种量，酸，碱和进水软管。 </li><li>在121°C高压灭菌20分钟的反应堆。 </li><li>酸碱水库（2 M盐酸，氢氧化钠2米）和相应的泵连接反应堆和设立的pH值5.0±0.05，pH值控制单元。 </li><li>连接反应堆的冷却水系统，并加热夹克各地的反应堆压力容器。 </li><li>安装相应的控制单元温度传感器，并设立了37个温度±0.1°C的温度控制单元。 </li><li>适合搅拌电机之上反应堆，并把服务与搅拌速度为200分钟-1。 </li><li>最小的无菌培养基中11槽添加无菌进水软管（250毫升）。为中等准备所有组件在121℃高压灭菌20分钟，混在了一起。三栽培的组件包括滑石粉（1 g / L的浓度3MgO•4SiO 2·H 2 O）（反应堆2），3 g / L的（反应堆）和10 g / L的（反应堆）。在使用之前，重新挂起微粒在50毫米醋酸钠缓冲液（pH值6.5），并把它添加到无菌培养基。在控制文化（无颗粒）微颗粒悬浮液取代50毫米醋酸钠缓冲液（pH值6.5）。 </li><li>新增的孢子悬浮液（接种）准备投入Wucherpfennig等。人（2011）11槽无菌进水软管（50毫升）的之一，使1X10 6毫升-1孢子的浓度。接种标志着开始种植（H = 0 H）。 </li><li>启动速度的1.0升分钟-1曝气。 </li></ol> 2。无菌采样后24，48和栽培72小时<ol><li>成flacon管50毫升的无菌培养液。 </li><li>用于测定生物量干重，β-呋喃果糖苷酶的活性和微观分析的样本。 </li></ol> 3。测定生物量干重的24后，48和栽培72小时<ol><li>生物样品要至少一式两份。 </li><li>重量与微观尺度的纤维素过滤器后，在干燥器和干燥过滤器放置在一个连接的水喷射真空泵的布氏漏斗。 </li><li>过滤定义的样本量（如10毫升）ND用10 mL去离子水冲洗过滤器，从生物中取出介质的化合物。 </li><li>皱纹的过滤器，一旦在中间，放置在一个玻璃培养皿和投入舱室干衣机，直到体重恒定（至少24小时）。 </li><li>在干燥器中冷却过滤器，并测量体重。 </li><li>作为过滤器的重量之间的差异并没有干生物量除以所使用的样本量计算生物量干重。 </li></ol> 4。神/ POD-文章胞外β-呋喃果糖苷酶的活性测定，24后，48和栽培72小时与他们一起工作时存储在冰样本不断。 <ol><li>过滤器1.5毫升培养液按培养液用注射器通过过滤器进入反应管槽醋酸纤维素过滤。储存在-20°C，直到使用的反应管。 </li><li>反应混合物用20μL样品和添加200μL1.65米，0.05 M磷酸缓冲液（pH值5.4）溶解，启动反应葡萄糖蔗糖的蔗糖。出反应，至少在卡里复制。 </li><li>在40°C孵育反应混合物加热块为20分钟。停止反应，在95°C孵育10分钟加热块。冷却反应混合物由储存在冰和自旋向下13.000Ğ离心机10分钟在4°C冷凝水</li><li>为了帐户的pH值和温度依赖葡萄糖蔗糖裂解，开展使用20μL去离子水，而不是20μL样品空白值。 </li><li>要交代的培养液中的残糖，开展每个样品为阴性对照。为此，使用20μL的样品和灭活β-呋喃果糖苷酶在95℃加热10分钟前添加蔗糖和孵化步骤4.3中所述。 </li><li>稀释样品，例如，下面的测量ured吸附在标定值范围内。 </li><li>开展一式三份酶检测。适用于微滴度板以及2μL稀释后的样品。申请使用10个不同的知情浓度（从1毫米到15毫米），而不是2μL样品10个葡萄糖溶液2μL每一个微滴度板的校准标准范围。对于零点校准，使用2μL去离子水，而不是2μL样品。 </li><li>加入200μL试剂解决方案，以每口井，通过使用多吸管。 </li><li>孵育的混合物在室温为10分钟。 </li><li>微滴度板存放在6°，直到测量（最多几个小时）， </li><li>在96日出微板读者和麦哲伦数据检索软件的使用450纳米测量的吸收。设置搅拌时间为5秒。和1秒的休息时间。 </li><li>打开电子表格的结果图表和使用范围的标准校准线建设: Ğlucose浓度=一个X吸收+ B </li><li>计算活动: 活动= X稀释倍数的空白值（吸收）×A + B </li><li>通过计算样品的活动和适当的阴性对照之间的差异，找出β-呋喃果糖苷酶的活性。 </li><li>计算，考虑到生物量干重和β-呋喃果糖苷酶的活性11以下使用特定的生产力。 </li></ol> 5。栽培72小时后镜和自动图像分析<ol><li> 3毫升左右，在塑料培养皿地方文化的暂停和他们，直到分离的形态结构与生理氯化钠溶液稀释。 </li><li>位于培养皿，在显微镜下，它的特点是集成或连接相机。收购和储存约100个，每个样品的形态结构图像（ 图2）。注意，对每个图像至少一个对象被完全描绘。 </li><li>打开图像处理程序ImageJ的12对同一样品的所有图像。将图像转换为黑色和白色在使用过程中工具的"二进制"（ 图2）。申请命令，以全系列的图像，使用宏的代码如下。 运行（，二"） </li><li>打开ImageJ的再次加工的二进制图像。计算直径，形状因子FERET的投影面积，周长，圆，长宽比，圆度为每一个分析工具"设置测量"的形象UND坚固。一个严重申请的命令ES图像使用如下的宏代码。 运行（"8位"）; （"二进制"）; 运行（"集规模..."，"距离= X已知= 1000像素= 1单位=微米全球"）; 运行（"集测量..."，"区外围形状FERET的限制显示重定向=无小数= 3"）; （"分析粒子的......"，"大小= 10000无限圆= 0.00-1.00展=大纲显示"）; 决定兴建一条直线横跨比例尺与1000微米的像素数量X。关联的直线比例尺长度在微米的像素数目。 </li><li>打开的结果图表，包含形状因子值，为每一个电子表格的形象。计算如下为每个图像的形态数量。 <img alt="式（1）" src="/files/ftp_upload/4023/4023eq1.jpg" /></li><li>计算平均值和标准偏差为形态，考虑到一个样品的所有图像。 </l我&gt; <li>使用与特定的生产力形态的图形相关的图形和数据分析程序，并确定由数学回归的数学关系。 </li></ol> 6。代表结果除了 ​​通过滑石粉微粒A.尼日尔 SKAN 1015形态是一个真正的颗粒形态从分散甚至菌丝的形态改变。而在标准条件下表现出颗粒形态，菌丝形态与滑石微粒的10克/升（ 图4）。补充介质创建。同时β-呋喃果糖苷酶的活性增加约3倍3-5。一个补充的1或3克/ L的滑石粉导致分散的形态，用一倍的呋喃果糖苷酶的活性（ 图4）。 由铁道部，微观粒子的依赖形态可以全面描述phology数可以使用自动图像分析测定的参数计算。浑圆，光滑的小球会出现在显微图像完美的圆圈。对于这样的颗粒形态数量值1。菌丝形态的最小片段，可以简化为一维的形态产生了0号线。因此，所有像拉长的不规则颗粒或团块的中间形态的形式，将有0和1之间的值。高，真菌粒子具有较大的表面或拉长颗粒在一个相当低的形态11号，将导致相当大的颗粒。 在标准条件下反应堆1形态展品约0.8形态。约0.1锰的形态特征与滑石粉10 g / L的4反应堆。反应堆2和3与1和3 g / L时，滑石粉的浓度，形态介于这两个极端，展现分散形态logy。由于微观粒子的依赖形态是β-呋喃果糖苷酶生产力密切相关， 图5类似的形态数量和生产力的数学关系得到。 <img alt="图1" src="/files/ftp_upload/4023/4023fig1.jpg" /> 答:1。实验设计和分析过程的总体方案。 尼日尔是培养（带或不带微颗粒）在72小时搅拌式生物反应器3的大号。 24后，48和72公顷样品测定生物量干重和β-呋喃果糖苷酶的活性，而这又是用于计算的具体生产力。 48小时后，真菌形态学检查显微镜和数字图像分析为特色。相关参数的图像分析相结合，一个形态，这是数学与特定的生产力相关。 <imgALT ="图2"的src ="/ files/ftp_upload/4023/4023fig2.jpg"/&gt; 从 A微观形态结构生成的图像图像处理步骤图2。 尼日尔 。步骤1:图像采集用显微镜。第2步:如果有必要的形象得到改善。步骤3:图像二值化，黑色和白色（二进制）在ImageJ的图像。步骤4:二进制图像处理和清除不必要的对象。第5步:ImageJ的开源计划"分析粒子"功能进行形态分析。 <img alt="图3" src="/files/ftp_upload/4023/4023fig3.jpg" /> 图3。 答:不同形态的形式尼日尔对增值微粒浓度依赖性。随着微粒浓度的增加，颗粒大小可精确下降小核壳小球，小羊群，甚至自由分散的菌丝。砷的形态工程pergillus尼日尔SKAn1015深层培养的微观粒子的补充。没有微粒（一），10毫克/升（B）0.1 g / L的（C）0.2 g / L的（D）0.3 g / L的（E）为0.6 g / L（女），1.0克/升（G）1.5 g / L的（H）2.0 g / L的（一），（十）2.5克/ L，3.0 g / L的（金），3.5克/升（L），4.0克/升（男克/升），4.5（N）5.0 g / L的（O）10克/ L，（P），15 g / L的（Q）20克/升（R）为30 g / L（）和40-50克/升（T）培养72小时后光镜图像。 <img alt="图4" src="/files/ftp_upload/4023/4023fig4.jpg" /> 图4。呋喃果糖苷酶的活性依赖的滑石粉微粒浓度为1 g / L的（反应堆），3 g / L的（反应堆）和10 g / L的（反应堆）。反应堆1辅以微颗粒;这里种植的标准条件下进行。 <img alt="图5" src="/files/ftp_upload/4023/4023fig5.jpg" /> 图5。代表良好的相关性形态（R 2 = 0.91）数量和具体的生产力。绘制的形态数量（横坐标）对特定生产率（坐标）。非线性回归得到指数的相关性。

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我什么都没有透露。

几十年来一直在生物技术的兴趣，因为真菌形态的修改。不同的研究都试图改变选定的工艺参数，如pH值，输入功率，温度，营养中等或接种浓度为1，但苦于相当不精确的和不完整的形态，控制高能源成本，抑制效果或产品不稳定，相比之下，微粒的补充，可以通过微调粒度和浓度变化的真菌形态的精密工程。这将打开新的可能性，以用于优化和度身订造的高产形态设计与A.生物技术生产的微型颗粒尼日尔和其他丝状微生物。 数字图像分析是一种简单的重复的方法来描述真菌的宏观形态。然而，各种大小，形状和表面字符的参数形态结构之三在文献中描述的真菌形态复杂的快速评估。相关参数的组合形态，避免了这方面的不足，不仅可以全面表征形态结构，也为数学与生产力直接相关。这再次呈现形态和过程形态的定制生产力的估计需要。 使用形态的号码，它是可以区分不同的沉淀和聚集形态4,5。为进一步发展形态数量的分形维数的审议似乎被看好。一个分形维数，给出了一个对象的填充属性13的复杂性和质量的测量，因此整体菌丝形态的表征豫定。 铬然而，eation一个高产的菌丝形态，可能会导致与工艺性能，尤其是在大规模种植的问题，菌丝生长形式，因为以前一直表现出更大的培养液粘度2。这将导致与传热传质问题，并形成停滞的非混合区，这需要更高的输入功率，使种植更昂贵的操作1。因此真菌的形态和培养液粘度之间的关系应被视为改变形态和进一步车型纳入。

customization of aspergillus niger morphology through addition of talc micro particles

丝状真菌A.尼日尔是一个在范围广泛，从食品到医药行业的工业生产过程中广泛使用的应变。这种丝状生物体中最引人注目的，往往不可控的特点之一是其复杂的形态。它的范围从密集的球形颗粒，粘稠的菌丝体（ 图1）。各种工艺参数和配料是众所周知的影响真菌的形态1。由于最佳生产力与具有特定形态的形式强烈，真菌形态，往往代表着生产力的工业生产中的瓶颈。 一个直线前进和优雅的方法，能够精确地控制形态形状是另外无机的不溶性微粒（如水合硅酸镁，三氧化二铝或钛硅酸盐氧化物）的培养基，有助于增加酶生产2-6。既然有1 OBV白条之间的依赖微观粒子的形态和产酶的相关性，这是可取的数学链接生产力和外观形态。因此，精确的定量和整体形态描述的目标。 因此，我们提出了一个方法来生成和表征微观粒子的依赖形态结构和相关真菌的形态与生产力（图1），这可能有助于更好地理解的丝状微生物形态。 重组菌株A。尼日尔 SKAn1015 3大号搅拌槽反应器中培养72小时。除了滑石粉微粒浓度1克/ L，3 g / L的10 g / L的接种前要通过各种不同的形态结构，再现产生。无菌抽取样本后24，48和72小时的成长进步和产生的酶的活性测定。 "形成的产品是高附加值的酶β-呋喃果糖苷酶，糖在食品或制药行业，其中除其他催化反应的蔗糖，葡萄糖7-9新形成的一个重要的生物催化剂。因此，量化后加入蔗糖葡萄糖意味着产β-呋喃果糖苷酶量。葡萄糖定量是由一个神/过氧化物酶的含量10，这是在96孔微滴度板的高通量分析修改。 72小时后真菌形态显微镜检查和数字图像分析为特色。这样做，宏真​​菌形态像FERET的直径，投影面积，周长，圆，长宽比，圆度和坚固的粒子形状因子的计算与开源图像处理程序ImageJ的。相关参数相结合，一个无量纲的形态数量（锰）11，从而使一个全面的表征真菌的形态。强调数学回归的形态数量和生产力密切相关。

Watch this Scientific Journal Video about Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles at JoVE.com

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

一个方法来精确地生成和全面表征丝状菌形态<em&gt;黑曲霉（Aspergillus niger）</em&gt;描述，它允许的外观形态和生产力的数学相关。

定制黑曲霉（Aspergillus niger）通过添加滑石粉微粒形态

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most ...

customization-aspergillus-niger-morphology-through-addition-talc

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

19.1K 次观看.  Technische Universität Braunschweig. 一个方法来精确地生成和全面表征丝状菌形态黑曲霉（Aspergillus niger）描述，它允许的外观形态和生产力的数学相关。

视频: Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae子囊孢子生产和收集微阵列实验。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

科研

生物学

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

环境科学

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

生物化学

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

A direct liquid-culture (DLC) screening method has been developed which significantly reduces the time required for polyethylene glycol (PEG)-mediated protoplast transformation of the filamentous fungus, Aspergillus oryzae, when employed for evaluation of the secretory production of heterologous proteins. This method dramatically increases the throughput of the evaluation protocol.

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A ...

遗传学

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

使用 SAP-AS 培养丛枝菌根真菌以增强观察

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the rhizosphere. Typically, AM fungi are cultured under in vivo conditions in pot culture with an autotrophic host or under in vitro conditions with Ri Transfer-DNA transformed roots (heterotrophic host) in a Petri dish. Additionally, the cultivation of AM fungi in pot culture occurs in an opaque and non-sterile environment. In contrast, in vitro culture involves the propagation of AM fungi in a sterile, transparent environment. The superabsorbent polymer-based autotrophic system (SAP-AS) has recently been developed and shown to combine the advantages of both methods while avoiding their respective limitations (opacity and heterotrophic host, sterility). Here, we present a detailed protocol for easy preparation, single spore inoculation, and observation of AM fungi in SAP-AS. By modifying the Petri dishes, high-resolution photographic and video observations were possible on living specimens, which would have been difficult or impossible with current in vivo and in vitro techniques.

作者聚焦：使用 SAP-AS 加强 AM 真菌研究 — 一种用于单孢子培养的新技术

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

Arbuscular mycorrhizal (AM) fungi are difficult to manipulate and observe due to their permanent association with plant roots and propagation in the ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

生物工程

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

定制黑曲霉（Aspergillus niger）通过添加滑石粉微粒形态

摘要

探索更多视频

此视频中的章节

Gibberella zeae子囊孢子生产和收集微阵列实验。

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特亨蒂埃拉阿曼尼西斯

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

数百个 C.白化病细胞生物原子力显微镜测量自动化

低温电子显微镜样品制备借助于聚焦离子束

定制黑曲霉（Aspergillus niger）通过添加滑石粉微粒形态

摘要

探索更多视频

此视频中的章节

Gibberella zeae子囊孢子生产和收集微阵列实验。

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

在基于高吸水性聚合物的自养系统上接种和观察丛枝菌根培养物

数百个 C.白化病 细胞生物原子力显微镜测量自动化

低温电子显微镜样品制备借助于聚焦离子束

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特亨蒂埃拉阿曼尼西斯

数百个 C.白化病细胞生物原子力显微镜测量自动化