Abstract：
The fastest replicating bacterium Vibrio natriegens is a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential of V. natriegens and collectively expand upon its utility as an emerging model organism for synthetic biology.
OBJECTIVE: Vibrio natriegens is an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of the V. natriegens model system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing \"clean\" derivative cells. Finally, we show the full extent of this technique\'s capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to the Vibrio community and specifically to the advancement of V. natriegens as a model system.
OBJECTIVE: Vibrio natriegens is an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of the V. natriegens model system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing \"clean\" derivative cells. Finally, we show the full extent of this technique\'s capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to the Vibrio community and specifically to the advancement of V. natriegens as a model system.
摘要：
复制最快的纳氏弧菌是分子和生物技术研究的新兴主力，具有有效的遗传操作工具。这里,我们通过鉴定中性插入位点,并展示了如何在该位点将两个选择标记交换为连续的自然转化轮次,从而扩展了通过自然转化(MuGENT)进行多重基因组编辑的能力.第二,我们证明了MuGENT可以通过在异位染色体位点插入基因来进行互补。此外,我们开发了一种强大的方法来治愈诱导自然转化所需的能力质粒。最后,我们证明了MuGENT产生大量缺失的能力;本研究中产生的280kb缺失是在单轮细菌靶向诱变中构建的最大的人工缺失之一.这些方法均提高了V.natriegens的遗传潜力，并共同扩展了其作为新兴的合成生物学模型生物的实用性。
目的：纳氏弧菌是一种新兴的分子和生物技术应用模式生物。它的快速增长，代谢多功能性，和易于遗传操作为合成生物学提供了理想的平台。这里,我们开发和应用新的方法来扩展V.natriegens模型系统的遗传能力。先前的研究开发了一种在单个步骤中操纵染色体多个区域的方法。这里,我们提供了使这种方法的效用多样化的新资源。我们还提供了一种技术，一旦操作完成，就可以从细胞中删除所需的遗传工具，从而建立“干净”的衍生细胞。最后,通过产生文献中报道的最大的染色体缺失之一，我们显示了该技术的全部能力。总的来说，这些新工具将广泛有益于弧菌群落，特别是作为模型系统的V.natriegens的发展。
目的：纳氏弧菌是一种新兴的分子和生物技术应用模式生物。它的快速增长，代谢多功能性，和易于遗传操作为合成生物学提供了理想的平台。这里,我们开发和应用新的方法来扩展V.natriegens模型系统的遗传能力。先前的研究开发了一种在单个步骤中操纵染色体多个区域的方法。这里,我们提供了使这种方法的效用多样化的新资源。我们还提供了一种技术，一旦操作完成，就可以从细胞中删除所需的遗传工具，从而建立“干净”的衍生细胞。最后,通过产生文献中报道的最大的染色体缺失之一，我们显示了该技术的全部能力。总的来说，这些新工具将广泛有益于弧菌群落，特别是作为模型系统的V.natriegens的发展。
