Principal Investigator Gregory Stephanopoulos
Effective optimization and analysis of genetic and metabolic networks relies on the development of broad and powerful molecular biological tools. Unlike gene knockouts, the task of gene overexpression must address the issue of expression level optimization. Furthermore, the optimal gene expression level can vary for each gene of interest. Therefore, a large dynamic range in gene expression is required to obtain a wide range of phenotypes. Often, these modulations are conducted through the tedious manipulation of variables such as plasmid copy number, induction conditions, and promoter type. These steps are required since a broad-range, functional tool for easily creating a dynamic range of constitutive gene expression is not available. To address this issue, a promoter library based on the bacteriophage PLTetO1 promoter was developed by creating a mutant library through the use of error-prone PCR. The promoters in this library were then screened and evaluated through flow cytometry based on the expression of a green fluorescent protein (GFP) reporter protein. From these sorted expression levels, a functional promoter library was created containing over 20 promoters for which GFP levels span 120 fold range of fluorescence. This wide dynamic range of promoter strengths allows for the optimization of gene expression levels. Here, we present our results on the analysis of these promoters on the basis of their sequences, transcriptional profiles, and the resulting phenotypic behavior. Furthermore, we address possible applications in quantitative functional genomics facilitated by this new tool.