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T4 DNA Ligase

T4 DNA连接酶

Company: New England Biolabs
Catalog#: M0202S
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Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor
Author:
Date:
2018-08-20
[Abstract]  Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of P. tricornutum is that the current selection methods for P. tricornutum transformants depend on the use of a limited number of antibiotic resistance genes. An alternative and more cost-effective selection method would be to generate auxotrophic strains of P. tricornutum by knocking out key genes involved in amino acid biosynthesis, and using ... [摘要]  硅藻是一种具有重要生态意义的真核微藻类,其特性使其对生物燃料,食品,化妆品和药品等生物技术应用具有吸引力。 Phaeodactylum tricornutum 是具有确定培养条件的模型硅藻,但缺乏简单而强大的遗传工具阻碍了常规遗传操作。有效设计 P的一个障碍。 tricornutum 是 P的当前选择方法。 tricornutum 转化体依赖于使用有限数量的抗生素抗性基因。另一种更具成本效益的选择方法是产生 P的营养缺陷型菌株。通过敲除参与氨基酸生物合成的关键基因,并使用基于质粒的生物合成基因拷贝作为选择标记,使三角酵母。以前关于 P基因敲除的研究。 tricornutum 使用biolistic转换将CRISPR-Cas9系统传递到 P.藻。非复制质粒的生物射弹转化可导致对 P的不期望的损伤。由于转化的DNA随机整合到基因组中,tricornutum 。随后固化编辑的细胞以防止Cas9的长期过表达是非常困难的,因为目前没有方法来切除整合的质粒。该协议采用新方法将Cas9或TevCas9系统传送到 P. tricornutum 通过来自细菌供体细胞的质粒的缀合。该过程涉及:1)设计和插入靶向 P的guideRNA。将tricornutum 尿素酶基因导入TevCas9表达质粒,该质粒也编码转移的接合起点,2)将该质粒安装在含有含有接合机制的质粒(pTA-Mob)的大肠杆菌中, ...

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 ...

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