Changing Plasmid Copy Number by Mutation of the pMB1 Origin of Replication (LB209)

Abstract

We are developing Programmed Evolution as a system to optimize metabolic pathways in E. coli. The system allows introduction of variation in the regulatory elements of a genetic circuit encoding enzymes that control the desired orthogonal metabolism. Bacteria with variations that contribute best to metabolic output are selected through a fitness module. We reasoned that variation in plasmid copy number (PCN) of vectors containing the genetic circuit would affect metabolic output. We designed and cloned mutations in the pMB1 origin of replication to produce a change in PCN. We mutated the promoter regions of the RNA I and RNA II genes. PCN was measured by the use of Real Time Quantitative Polymerase Chain Reaction (RT‐qPCR) by comparison of five serial dilutions of chromosomal and plasmid DNA. Analysis of Ct values of plasmid and chromosomal DNA suggested altered PCN in some of the mutants. PCN measurements from RT‐qPCR were corroborated with measurements of DNA yield from plasmid preps of bacteria carrying the mutated origins. The results provide a collection of origins with several PCNs that can be used to introduce variation that is expected to affect metabolic output during Programmed Evolution.