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T4 DNA Ligase

T4 DNA连接酶

Company: New England Biolabs
Catalog#: M0202S
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Characterization of Protein Domain Function via in vitro DNA Shuffling
Author:
Date:
2018-06-05
[Abstract]  We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active hybrids were found to possess much fewer amino acid substitutions than the few hybrids that became inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. Certain important residues that played important functional roles in mediating enzyme activity were identified. These findings suggest that DNA shuffling is an effective ... [摘要]  我们最近研究了通过对CTX-M-14和EMX-M-14的片段进行DNA改组来驱动CTX-M型β-内酰胺酶进化的分子事件, bla CTX-M-15基因。 总共51种杂合酶的分析显示酶活性在大多数情况下可以保持,但是酶活性杂合体被发现比少数杂交体具有少得多的氨基酸取代,这表明构建体中的点突变而不是 两个靶基因片段的重新洗牌将更可能导致CTX-M活性的破坏。 确定了一些在介导酶活性中起重要作用的重要残基。 这些发现表明,DNA改组是一种有效的方法来鉴定和表征细菌蛋白质中的重要功能结构域。

【背景】DNA重组是一种自然过程,通过该过程,细菌之间交换遗传物质以增强环境压力下的生存适应性。几种杂交CTX-M-内酰胺酶(CTX-M-64,CTX-M-123,CTX-M-137和CTX-M-132)可能是由bla CTX-M-14和 bla CTX-M-15基因是世界上最常见的变异体,近年来已有报道(Nagano et al。 ,2009; Tian et al。,2014; He et al。,2015; Liu et。, 2015年)。在这些杂合酶中,包含CTX-M-15的N-和C-末端部分和CTX-M-14的中间片段的CTX-M-64显示出比其亲本原型更高的催化活性(He <等)。,2015)。

DNA改组是一种分子途径,被设计为通过PCR介导的两种靶基因的随机组合来模拟和加速进化过程(Crameri ...

Reduced Representation Bisulfite Sequencing in Maize
Author:
Date:
2018-03-20
[Abstract]  DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore ... [摘要]  DNA甲基化是调节植物发育的表观遗传修饰(Law and Jacobsen,2010)。全基因组亚硫酸氢盐测序(WGBS)是用单碱基分辨率分析全基因组甲基化模式的最先进的方法(Cokus et al。,2008)。然而,对于具有大基因组的生物体,例如玉米的2.1Gb基因组,WGBS可能非常昂贵。代表性亚硫酸氢盐测序(RRBS)已经在哺乳动物研究中发展(Smith等人,2009)。通过用大小选择范围大约40-220bp的 Msp 消化基因组,可以对仅涵盖〜1%人类基因组的CG富含区域进行特异性测序。然而,与哺乳动物基因组不同,植物基因组不显示清楚的CpG岛。因此原来的RRBS协议不适用于工厂。因此,我们开发了一种计算机管道来选择特定的酶以生成感兴趣区域(ROI) - 富集的,例如,富含启动子的,减少的植物表达基因组(例如, Hsu et al。,2017)。通过用MseI消化玉米基因组并选择40-300bp片段,我们测序了大约四分之一的玉米基因组,同时保留了84.3%的启动子信息。该协议已在玉米中成功建立,可广泛应用于任何基因组。我们的计算机管道系统与RRBS文库制备方案相结合,允许进行计算分析和实验验证。

【背景】DNA甲基化是一种可遗传的表观遗传修饰,通过调节基因表达和染色质结构在动物,植物和真菌的许多发育过程中发挥重要作用(Law and ...

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
Author:
Date:
2018-01-20
[Abstract]  With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid ... [摘要]  随着CRISPR / Cas9技术作为基因组编辑的标准工具的最近实施,全世界的实验室正在经历PCR以来分子生物学方面最大的进步之一。这种方法的关键优点是其简单和普遍适用于任何物种的门。特别感兴趣的是广泛研究的革兰氏阴性细菌大肠杆菌,因为它被认为是研究和工业用途的主力。在这里,我们提出了一个简单,强大和有效的协议,使用CRISPR / Cas9系统结合λ红色基因敲除机器。大肠杆菌。在我们的程序中最重要的是使用双链供体DNA和固化策略来去除导向RNA编码质粒,其允许在仅仅两个工作日后开始新的突变。我们的方案允许多个具有高诱变效率的基因敲除株,适用于高通量的方法。

【背景】革兰氏阴性细菌大肠杆菌是生物技术工程中最重要的生物之一。已在能源,农业,食品生产,生物技术,医药等不同行业的各种流程中成功实施。由于不断的技术进步,生物技术部门正在迅速发展。特别是,CRISPR / Cas9技术可能是PCR(分子)生物学最大的革命(Ledford,2015)。简而言之,CRISPR / Cas9保护细菌免受诸如质粒和病毒等侵入性遗传因子的影响(Marraffini,2015)。利用这种从原核生物获得的免疫系统,已经开发了基于CRISPR / Cas9系统的基因组编辑的非常有力的工具(Jinek等人,2012)。

CRISPR / ...

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