期刊
ACS SYNTHETIC BIOLOGY
卷 10, 期 7, 页码 1739-1749出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00138
关键词
CRISPR/Cas9; genetic tools; thermophile; stable plasmid expression; synthetic biology; curing plasmids
资金
- UK Biotechnology and Biological Sciences Research Council through CASE studentship [BB/L016478/1, BB/N022718/1]
- ATUM
- IB Catalyst grant MAXIBIO
This study expands on the genetic tools available for engineering the industrial chassis Parageobacillus thermoglucosidasius by assembling and testing a range of promoters, ribosome binding sites, reporter genes, and implementing CRISPR/Cas9 genome editing based on two different thermostable Cas9 nucleases.
The relentless rise in the levels of atmospheric greenhouse gases caused by the exploitation of fossil fuel necessitates the development of more environmentally friendly routes to the manufacture of chemicals and fuels. The exploitation of a fermentative process that uses a thermophilic chassis represents an attractive option. Its use, however, is hindered by a dearth of genetic tools. Here we expand on those available for the engineering of the industrial chassis Parageobacillus thermoglucosidasius through the assembly and testing of a range of promoters, ribosome binding sites, reporter genes, and the implementation of CRISPR/Cas9 genome editing based on two different thermostable Cas9 nucleases. The latter were used to demonstrate that the deletion of the two native plasmids carried by P. thermoglucosidasius, pNCI001 and pNCI002, either singly or in combination, had no discernible effects on the overall phenotypic characteristics of the organism. Through the CRISPR/Cas9-mediated insertion of the gene encoding a novel fluorescent reporter, eCGP123, we showed that pNCI001 exhibited a high degree of segregational stability. As the relatively higher copy number of pNCI001 led to higher levels of eCGP123 expression than when the same gene was integrated into the chromosome, we propose that pNCI001 represents the preferred option for the integration of metabolic operons when stable commercial strains are required.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据