CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae
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Author:
Date:
2017-05-20
[Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four ...
[摘要] 染色体工程是应用于基础生物学和生物技术的重要技术。染色体分裂技术称为PCS(PCR介导的染色体分裂)已经被开发为发芽酵母中的基本染色体工程技术。然而,PCS技术的分割效率不够高,不能一次实现多次分割。该协议描述了通过称为CRISPR-PCS的新技术在芽状酵母酿酒酵母中实现同时和多个染色体分裂的过程。基因组中至少四个独立的位点可以通过一次转化来分裂。与常规PCS技术相比,获得多重分裂酵母菌株的总时间和劳动力大大降低。
背景 能够快速有效地操纵多个遗传基因座或染色体区域的染色体工程技术变得越来越重要。这些技术为阐明染色体和基因组功能提供了有力的手段。此外,它可以用于通过创建广泛的遗传变体来繁殖有用的菌株。在芽殖酵母酿酒酵母(Saccharomyces ...
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Pseudomonas syringae Flood-inoculation Method in Arabidopsis
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Author:
Date:
2017-01-20
[Abstract] Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings grown on one-half strength MS medium. We also describe a method to evaluate the bacterial growth in Arabidopsis.
[摘要] 丁香假单胞菌 pv。 由于其在拟南芥中的致病性,已经将用于番茄细菌斑病的番茄株DC3000( DC3000)用作模型病原体 >。 在这里,我们展示了一种基于在一半强度MS培养基上生长的幼苗拟南芥幼苗的快速可靠的接种方法。 我们还描述了一种评估拟南芥细菌生长的方法。
背景 A。 DC3000模型病理学被广泛用于研究微生物发病机制和植物先天免疫的分子机制(Ishiga等,2012和2016; Ishiga和Ichinose,2016)。 虽然已经开发了几种接种方法来研究该模型系统中的相互作用,但迄今报道的方法之一与自然界中发生的方法相似。
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Analysis of L- and D-Amino Acids Using UPLC
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Author:
Date:
2014-09-05
[Abstract] With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in ...
[摘要] 除了甘氨酸,α-氨基酸具有光学活性,并且可以形成每个氨基酸的两种光学异构体(L-和D-)。分析技术的最新发展表明,几种游离D-氨基酸如D-天冬氨酸,D-丝氨酸和D-丙氨酸存在于包括人在内的许多种生物中,并且具有生物学上的重要作用。 D-天冬氨酸调节动物和人的生殖活性。 D-丝氨酸作为Nε-甲基-D-天冬氨酸受体的共激动剂,其介导谷氨酸能神经传递。 D-丙氨酸在甲壳类动物和软体动物中起到类似渗透物的作用。在该协议中,我们描述了使用超高效液相色谱(UPLC)分析L-和D-氨基酸的方法。为了分析D-和L-氨基酸,首先将对映异构体转化为非对映异构体(非对映异构体是与物镜和镜像无关的立体异构体,并且不是对映异构体),使用前柱衍生化,邻苯二甲醛加上Nε - 酰化的半胱氨酸(Nε - 乙酰基-L-半胱氨酸或Nε - 叔丁基氧羰基-L 。所得衍生物是荧光非对映异构体。然后使用UPLC在十八烷基硅烷基固定相上分离所得荧光异吲哚衍生物,并通过包括在UPLC系统中的荧光检测器检测荧光。使用该方法,可以分析16种D-氨基酸。
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