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Mineral oil

矿物油

Company: Sigma-Aldrich
Catalog#: M5904
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Bacterial Microcolonies in Gel Beads for High-throughput Screening
Author:
Date:
2018-07-05
[Abstract]  High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.

Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies ...
[摘要]  在细菌群体中表达的DNA文库的高通量筛选用于鉴定显示感兴趣性质的潜在稀有成员是在许多实验中成功的关键步骤,例如蛋白质和合成回路的定向进化以及用于鉴定增益的深度突变扫描 - 或功能丧失的突变体。

在这里,我描述了一种用于高通量筛选凝胶珠中细菌(大肠杆菌)微菌落的方案。将单细胞包封成用微流体装置产生的单分散油包水乳液液滴。水溶液还含有琼脂糖,其在冰上冷却时凝胶化,从而在液滴内部形成固体凝胶珠。在乳液温育期间,细胞在珠内生长成单克隆微菌落。在从乳液中分离凝胶珠并通过荧光激活细胞分选(FACS)分选后,从凝胶珠中回收细菌,然后准备进行进一步的分选,诱变或分析。为了通过FACS分类,该方案需要荧光读数,例如荧光报告蛋白的表达。测量微小菌落的平均荧光信号降低了高表型细胞间变异性的影响,并且与单细胞分选相比提高了灵敏度。我们应用这种方法在ON和OFF状态下对pBAD启动子文库进行分类(Duarte et al。,2017)。

【背景】荧光激活细胞分选(FACS)具有> 10 7 事件/ h的无与伦比的筛选通量(Davies,2012)。然而,通过FACS根据其荧光分选单个细胞以筛选合成回路的文库(Schaerli和Isalan,2013)经常受到高表型细胞间变异性的阻碍。或者,可以对水凝胶珠中所含的小细胞集落(微集落)进行分类(Weaver ...

Xenopus laevis Oocytes Preparation for in-Cell EPR Spectroscopy
Author:
Date:
2018-04-05
[Abstract]  One of the most exciting perspectives for studying bio-macromolecules comes from the emerging field of in-cell spectroscopy, which enables to determine the structure and dynamics of bio-macromolecules in the cell. In-cell electron paramagnetic resonance (EPR) spectroscopy in combination with micro-injection of bio-macromolecules into Xenopus laevis oocytes is ideally suited for this purpose. Xenopus laevis oocytes are a commonly used eukaryotic cell model in different fields of biology, such as cell- and development-biology. For in-cell EPR, the bio-macromolecules of interest are microinjected into the Xenopus laevis oocytes upon site-directed spin labeling. The sample solution is filled into a thin glass capillary by means of Nanoliter Injector and after that ... [摘要]  研究生物大分子的最令人兴奋的观点之一来自于新兴的细胞内光谱学领域,它能够确定细胞中生物大分子的结构和动力学。细胞内电子顺磁共振(EPR)光谱结合将生物大分子微注射到非洲爪蟾卵母细胞中非常适合于此目的。非洲爪蟾卵母细胞是生物学不同领域常用的真核细胞模型,如细胞和发育生物学。对于细胞内EPR,感兴趣的生物大分子通过定点自旋标记显微注射到非洲爪蟾卵母细胞中。通过Nanoliter注射器将样品溶液填充到薄玻璃毛细管中,然后通过小心地穿刺薄膜将其微注射入非洲爪蟾卵母细胞的黑色动物部分。之后,取决于最终的细胞内EPR实验的种类,将三个或五个显微注射的非洲爪蟾卵母细胞装载到Q波段EPR样品管中,随后进行任选的休克冷冻(用于实验冷冻溶液)并且在期望的温育时间之后测量(在低温或生理温度下)。由于显微注射样品的细胞毒性作用和顺磁性自旋标记在还原性细胞环境中的稳定性,孵育时间受到限制。通过监测细胞形态和减少动力学来量化这两个方面。

【背景】电子顺磁共振(EPR)光谱学是用于表征顺磁系统的选择方法(Atherton,1993; Gerson等人,1994; Jeschke和Schweiger,2001)。反磁性生物大分子可以通过定点自旋标记(SDSL)进行EPR光谱学分析,通常使用氮氧化物作为自旋标记(Hubbell和Altenbach,1994; Feix和Klug,2002; ...

Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes:Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor
Author:
Date:
2017-01-20
[Abstract]  Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways. This protocol has been used to study plant ion channels, including the SLAC1 channel (SLOW ANION CHANNEL-ASSOCIATED 1), in particular SLAC1 activation by either the protein kinase OST1 (OPEN STOMATA 1), Ca2+-dependent protein kinases (CPKs) or the ... [摘要]  在非洲爪蟾卵母细胞中记录的双电极电压钳(TEVC)提供了一种强大的方法来研究离子通道蛋白的功能和调节。这种方法提供了一种众所周知的用于表征在非洲爪蟾卵母细胞中表达的离子通道或转运蛋白的工具。卵母细胞的质膜由两个微电极引起,一个用于电压感测,另一个用于电流注入。在这里,我们列出了一个允许多组分信号通路强制重组的协议。该方案已经用于研究植物离子通道,包括SLAC1通道(SLOW ANION CHANNEL-ASSOCIATED 1),特别是SLAC1通过蛋白激酶OST1(OPEN STOMATA 1),Ca 2 +依赖性蛋白激酶(CPK)或GHR1(GUARD细胞过氧化氢抗性1)跨膜受体样蛋白。显示了通过“单体”ABA(脱落酸)受体RCAR1 / PYL9(PYRABACT INRESISTANCE1 [PYR1] / PYR1-样[PYL] / ABA受体[RCAR]的调节因子]重建SLAC1阴离子通道的脱落酸活化的数据。通过共表达脱落酸信号核心的四个组分。该方案也适用于研究其他离子通道功能和调节机制,以及转运蛋白。背景 ...

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