Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA
Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA.
Appl Environ Microbiol. 2018 Jul 2;84(14). doi: 10.1128/AEM.00850-18. Print 2018 Jul 15.
is used as a model for a number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multistep process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the chromosome without cloning. We developed a uniform approach for generating deletions using PCR splicing by overlap extension (SOEing) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT (FLP recognition target) sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome for purposes such as complementation. Finally, we generated derivatives of these tools to facilitate different applications, such as inducible gene expression and assessing protein production. We demonstrated the utility of these tools by deleting and inserting genes known or predicted to be involved in motility. While developed for strain ES114, we anticipate that these tools can be adapted for use in other strains and, potentially, other microbes. is a model organism for studying a variety of important processes, including symbiosis, biofilm formation, and quorum sensing. To facilitate investigation of these biological mechanisms, we developed approaches for rapidly generating deletions and insertions and demonstrated their utility using two genes of interest. The ease, consistency, and speed of the engineering is facilitated by a set of antibiotic resistance cassettes with common linker sequences that can be amplified by PCR with universal primers and fused to adjacent sequences using splicing by overlap extension and then introduced directly into , eliminating the need for cloning and plasmid conjugation. The antibiotic cassettes are flanked by FRT sequences, permitting their removal using Flp recombinase. We augmented these basic tools with a family of constructs for different applications. We anticipate that these tools will greatly accelerate mechanistic studies of biological processes in and potentially other species.
被用作许多过程的模型，包括共生、群体感应、生物发光和生物膜形成。许多这些研究依赖于生成缺失突变体并对其进行补充。然而，工程这些菌株是一个耗时的、多步骤的过程，依赖于克隆和亚克隆。在这里，我们描述了一组工具，可以在不进行克隆的情况下快速工程染色体中的缺失和插入。我们开发了一种使用抗生素盒侧翼的标准化接头序列通过 PCR 拼接重叠延伸 (SOEing) 生成缺失的通用方法。将盒 SOEing 到目标基因上下游的序列生成可以直接通过自然转化引入的 DNA 产物。选择引入的抗生素抗性标记物可在单个步骤中获得感兴趣的缺失。因为这些盒还包含抗性标记物侧翼的 FRT(FLP 识别靶标)序列，所以 Flp 重组酶可用于生成无标记的、框内缺失。我们开发了类似的方法和工具，用于在染色体上的良性位点快速插入特定基因，以进行互补等目的。最后，我们生成了这些工具的衍生物，以促进不同的应用，例如诱导基因表达和评估蛋白质生产。我们通过删除和插入已知或预测与运动性相关的基因证明了这些工具的实用性。虽然这些工具是为 ES114 菌株开发的，但我们预计这些工具可以适应其他 菌株的使用，并且可能适应其他微生物。是研究各种重要过程的模式生物，包括共生、生物膜形成和群体感应。为了促进对这些生物学机制的研究，我们开发了快速生成缺失和插入的方法，并使用两个感兴趣的基因证明了它们的实用性。一组具有通用接头序列的抗生素抗性盒的使用使工程的简便性、一致性和速度得到了提高，这些盒可以通过 PCR 用通用引物扩增，并通过重叠延伸拼接融合到相邻序列，然后直接引入 ，从而消除了克隆和质粒连接的需要。抗生素盒被 FRT 序列包围，允许使用 Flp 重组酶将其去除。我们用一组用于不同应用的构建体增强了这些基本工具。我们预计这些工具将极大地加速 和潜在其他 物种中生物学过程的机制研究。
