Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor
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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, ...
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Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes
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Author:
Date:
2016-12-05
[Abstract] Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors from plants constitute a valuable approach for the development of new therapeutic strategies. Herein we describe a simple and accessible microplate method for the study of the specific and dose-response carboxypeptidase A inhibitory activities present in Andean potato tubers. Our protocol combines an extraction method optimized for small protein inhibitors in plant tissues, with the measurement of enzyme kinetics using a ...
[摘要] 金属羧肽酶(MCP)是锌依赖性外肽酶,其催化蛋白质和肽中的C-末端酰胺键的水解。它们参与多种生理过程,并且最近作为生物医学中的相关药物靶标出现(Arolas等人,2007)。在这种情况下,从植物研究和发现新的MCP抑制剂构成了开发新的治疗策略的有价值的方法。在这里我们描述一种简单和可访问的微孔板方法研究特定和剂量反应羧肽酶A抑制活动存在于安第斯马铃薯块茎。我们的协议结合了针对植物组织中的小蛋白抑制剂优化的提取方法,使用酶标仪测量酶动力学。这些仪器能够在很短的时间内读取小样品体积,因此减少了高通量筛选实验的时间和成本。虽然本协议描述了安第斯土豆的研究,我们的方法也适用于分析其他植物样品。 关键字:金属羧肽酶,羧肽酶A,抑制剂,抑制活性,微孔板测定,土豆 [背景] 蛋白质蛋白酶抑制剂是作为其防御昆虫攻击的防御系统的一部分产生的伤口诱导分子(Graham等人,1981; Villanueva等人,1998)。在研究的抑制剂中,只有两种对MCP具有特异性,即,马铃薯羧肽酶抑制剂(PCI)及其在番茄植物(TCI)中发现的紧密同源物。在过去几十年中,Solanaceae中MCP抑制剂的存在已被广泛报道,揭示马铃薯(马铃薯)作为MCP抑制剂的最重要来源之一(Hass等人, ,1979;Obregón等人,2012; ...
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