| 摘要 |
Described herein is the development of a multi-plasmid system (Compatible Antibiotic-free Multi-Plasmid System, CAMPS) for the expression of one or more nucleic acid sequences of interest in specially engineered host cells and grown in antibiotic-free medium. A panel of compatible plasmids was engineered in which each plasmid comprises an identical origin of replication (Ori) which only differs between plasmids in the loop sequence/s of the RNA I/II region of the Ori. Thus, these plasmids share the same replication mechanism but vary in copy numbers. In order to maintain these multiple plasmids without using antibiotics, multiple conditional essential genes (CEG) from the host genome were grafted into these plasmids. As a result, all of these co-existing plasmids carrying the CEG were maintained in a host where the corresponding CEGs were knocked out from the host's genome during fermentation. Said CAMPS system has broad utility in metabolic engineering and synthetic biology. |
