Deb Shalini S, Reshamwala Shamlan M S, Lali Arvind M
DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India.
DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India.
J Microbiol Methods. 2016 Jun;125:49-57. doi: 10.1016/j.mimet.2016.04.006. Epub 2016 Apr 9.
Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated.
代谢工程策略通常采用多拷贝附加体质粒来过表达基因。然而，基于染色体的过表达更受青睐，因为它避免了使用选择压力并减轻了细胞的代谢负担。我们构建了一系列用于λ Red介导的大肠杆菌基因组工程的模板质粒。这些模板质粒允许构建基因组整合盒，可用于在预定位点整合单拷贝的DNA序列或替换启动子区域。构建的盒式结构在实现的表达水平和用于选择的抗生素方面提供了灵活性，同时还允许构建无标记菌株。模板质粒的模块化设计允许替换遗传元件以构建新的模板。文中展示了使用模板质粒进行基因整合和启动子替换的过程。
