Multiplex Plasmid Engineering (MPE) for Fine Tuning the Expression Level of Red Fluorescent Protein

摘要

Many applications of metabolic engineering need balanced expression of several genes in synthetic metabolic pathways and genetic circuits. But it is often difficult to achieve the best expression level for all the genes for a desired function due to the complexity of biological systems. Here, we present Multiplex Plasmid Engineering (MPE) for high efficient tuning the expression level of genes in plasmids. First, red fluorescent protein (rfp) was tested to be utilized as a reporter gene for assaying recombineering efficiency on plasmids. Then ssDNA and plasmid co-transformation technique was exploited to achieve 25 % recombineering efficiency which is compatible to genome recombination. At last, as a proof of concept experiment, MPE was applied to tune the expression level of rfp gene and successfully generated dispersed distribution of rfp expression level. Our method should potentially be used for simultaneously tuning the expression level of several genes in metabolic pathways.