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Archaea, a domain of single-celled microorganisms, are classified into five major phyla based on genetic and biochemical characteristics: Euryarchaeota, Crenarchaeota, Thaumarchaeota, Korarchaeota, and Nanoarchaeota. Among these, the phylum Euryarchaeota is notable for its remarkable diversity in morphology, metabolism, and ecological adaptations.Morphological and Metabolic DiversityMembers of Euryarchaeota exhibit a variety of cellular shapes, including rods and cocci. Their metabolic pathways...
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Psychology is the scientific discipline dedicated to understanding both observable behavior and the internal mental processes underlying such behavior. It aims to comprehend human nature and apply this understanding to solve practical problems, enhance well-being, and improve societal outcomes. An example of psychology's application is the study of prosocial behavior, such as why and under what conditions individuals might help strangers in need. This process involves describing observed...
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The concept of a cell started with microscopic observations of dead cork tissue by Robert Hooke in 1665. Hooke coined the term "cell" based on the resemblance of the small subdivisions in the cork to the rooms that monks inhabited, called cells. About ten years later, Antonie van Leeuwenhoek became the first person to observe the living and moving cells under a microscope. In the century that followed, the theory that cells represented the basic unit of life developed.
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Archaea, one of the three domains of life, exhibit remarkable diversity and adaptability, thriving in both extreme and moderate environments. Historically, most identified archaea have been classified into two major phyla: Euryarchaeota and Crenarchaeota. However, recent molecular studies have expanded this classification to include three additional phyla: Thaumarchaeota, Nanoarchaeota, and Korarchaeota, each exhibiting unique characteristics and ecological roles.Thaumarchaeota: Mesophiles...
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Excavata is a diverse group of protists that includes both chemoorganotrophic and phototrophic species, with some thriving in anaerobic environments. Among the key groups within Excavata are diplomonads and parabasalids, which are flagellated protists that lack mitochondria and chloroplasts. These microorganisms typically inhabit anoxic environments, such as the intestines of animals, where they exist either symbiotically or as parasites, relying on fermentation for energy production. Some...
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uSort-M:可扩展的用户定义的序列的分离,从不同的聚合图书馆.

Micah B Olivas, Patrick J Almhjell, Jack D Shanahan

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    概括

    我们开发了用户定义的Sorted Mutants (uSort-M) 来快速隔离和识别来自聚合库的蛋白质变体. 这种具有成本效益的方法加速了功能表征,弥合了蛋白质发现和理解之间的差距.

    科学领域:

    • 生物化学和分子生物学
    • 基因组学和蛋白质组学
    • 合成生物学 合成生物学

    背景情况:

    • 高通量测序和计算式蛋白质设计加速了蛋白质的发现,但超越了功能性特征.
    • 目前用于单个变异分析的方法受到基因合成高成本的限制,将研究限制在小变异子集.
    • 由于分析瓶,在识别新型蛋白序列和理解它们的功能之间存在很大的差距.

    研究的目的:

    • 开发一个具有成本效益和高效的工作流程,以从大型的,聚合的库中分离和识别单个蛋白质变体.
    • 为了克服昂贵的基因合成在大规模功能性蛋白质表征中所带来的局限性.
    • 通过高通量方法发现的各种蛋白质变体的快速功能注释.

    主要方法:

    • 开发了用户定义的Sorted Mutants (uSort-M),集成了聚合DNA合成,对大肠杆菌的自动细胞分类和长读序列.
    • uSort-M可以容纳通过各种方法生成的聚合库,包括多重组装,易发生错误的PCR和聚合站点定向突变发生.
    • 将单个细菌克隆自动分类到384个井板中,在1到2小时内为8个板实现了高效率 (高达90%的单克隆培养).

    主要成果:

    • 通过使用Sort-M.成功地从聚合的库中分离和识别了单个变体.
    • 应用到一个328个成员的扫描突变生成库中,可恢复96%的所需变体,成本比传统合成低五倍.

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  • 长读序列提供了可访问,快速和经济有效的序列识别,容忍图书馆多样性和片段长度变化.
  • 数字模拟证明了工作流的可扩展性,并预测了目标覆盖范围的抽样工作.

    • 结论:

    • uSort-M显著降低了从聚合图书馆中分离和测序单个蛋白质变体所需的成本和时间.
    • 工作流的通用性,效率和标准仪器的使用消除了对大规模蛋白质功能表征的关键障碍.
    • 这种方法有助于弥合蛋白质发现和功能注释之间的差距,加速生物研究.