Brumwell Stephanie L, Van Belois Katherine D, Nucifora Daniel P, Karas Bogumil J
Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
Department of Biology, The University of Western Ontario, London, ON, N6A 5B7, Canada.
Biodes Res. 2023 Mar 15;5:0009. doi: 10.34133/bdr.0009. eCollection 2023.
high resistance to various stressors combined with its ability to utilize sustainable carbon sources makes it an attractive bacterial chassis for synthetic biology and industrial bioproduction. However, to fully harness the capabilities of this microbe, further strain engineering and tool development are required. Methods for creating seamless genome modifications are an essential part of the microbial genetic toolkit to enable strain engineering. Here, we report the development of the SLICER method, which can be used to create seamless gene deletions in This process involves (a) integration of a seamless deletion cassette replacing a target gene, (b) introduction of the pSLICER plasmid to mediate cassette excision by I-I endonuclease cleavage and homologous recombination, and (c) curing of the helper plasmid We demonstrate the utility of SLICER for creating multiple gene deletions in by sequentially targeting 5 putative restriction-modification system genes, recycling the same selective and screening markers for each subsequent deletion. While we observed no significant increase in transformation efficiency for most of the knockout strains, we demonstrated SLICER as a promising method to create a fully restriction-minus strain to expand the synthetic biology applications of including its potential as an in vivo DNA assembly platform.
对各种应激源的高抗性及其利用可持续碳源的能力，使其成为合成生物学和工业生物生产中颇具吸引力的细菌底盘。然而，要充分发挥这种微生物的能力，还需要进一步的菌株工程改造和工具开发。创建无缝基因组修饰的方法是微生物遗传工具包中实现菌株工程改造的重要组成部分。在此，我们报告了SLICER方法的开发，该方法可用于在[具体微生物名称未给出]中创建无缝基因缺失。这个过程包括：（a）整合一个无缝缺失盒以取代目标基因；（b）引入pSLICER质粒，通过I-I内切酶切割和同源重组介导盒式切除；（c）去除辅助质粒。我们通过依次靶向5个假定的限制修饰系统基因，回收相同的选择和筛选标记用于后续的每次缺失，证明了SLICER在[具体微生物名称未给出]中创建多个基因缺失的实用性。虽然我们观察到大多数敲除菌株的转化效率没有显著提高，但我们证明了SLICER是一种有前景的方法，可用于创建一个完全无限制的菌株，以扩展[具体微生物名称未给出]的合成生物学应用，包括其作为体内DNA组装平台的潜力。
