Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering
Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering
复制标题
DOI：
10.1007/s10529-010-0455-x
复制
发表时间：
2011-03-01
影响因子：
2.7
通讯作者：
Smith, Hamilton O.
中科院分区：
文献类型：
--
作者：
Ramon, Adi;Smith, Hamilton O.
开发了用于将启动子和基因盒组合组装成生化途径的基于接头的方法。构建了一个含有144个组合的乙酸利用途径基因的合成文库，其中包括3个启动子和4个基因变体。杆菌将体外恒温组装的文库导入E. coli突变株acs-，pta-，ackA-，并选择用于恢复乙酸利用。81%的筛选的菌落含有完整的功能途径。分析了30个阳性克隆，占144个启动子-基因组合的10%。
A linker-based approach for combinatorial assembly of promoter and gene cassettes into a biochemical pathway is developed. A synthetic library containing 144 combinations, with 3 promoters and 4 gene variants, was constructed for the ackA and pta genes of the acetate utilization pathway in E. coli. The in vitro isothermal assembled library was then introduced into E. coli mutant (acs-, pta-, ackA-) and selected for restoration of acetate utilization. 81% of the colonies screened contained the complete functional pathway. Thirty positive clones were analyzed and accounted for 10% of the 144 promoter-gene combinations.