摘要：Deinococcus radiodurans' 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 D. radiodurans. 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-SceI endonuclease cleavage and homologous recombination, and (c) curing of the helper plasmid. We demonstrate the utility of SLICER for creating multiple gene deletions in D. radiodurans 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 D. radiodurans, including its potential as an in vivo DNA assembly platform.

摘要：这个事实我们知道了很久:一种生物,若遇到人为的物理、化学、生物方法引导,或者遗传因子得到重组,就可能在合适的新环境中发生多元突变。世界美术的古典风格、中世纪风格、文艺复兴风格、巴洛克风格,艺术设计的现代理念、后现代理念,手工艺的传统方式、"莫里斯方式"、"柳宗悦方式"……我们看到了,一部历史,就是传统秩序,经演进中的社会经济、政治、哲学、文化、生活方式等新生因子的扰动催化,最后产生复合变异的结果。