Rapid optimization of gene dosage in E. coli using DIAL strains

We engineered two sets of strains to constitutively provide a trans-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a constant level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein.The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways.Optimizing desired outcomes by varying a design parameter is a staple of almost every engineering field, from mechanical engineers tweaking blade angles on a wind turbine to civil engineers altering the timing of traffic lights. Similarly, genetic engineers alter gene expression levels to optimize some desirable phenotype. Strong overproduction of single proteins can impose a metabolic burden on E. coli, and often a lower expression level leads to improved phenotype [1]. In multi-subunit proteins and genetic circuits, expression of particular proteins often needs to be balanced for proper function (e.g. [2,3], and [4]). Extensive work has established methods for achieving expression of a gene or operon at a particular level, including control of transcription using standard promoter sets [5], modulation of RNA processing [6], and control of translation through ribosome binding site (RBS) manipulation [7]. However, using these tools to investigate the desired expression level of a single gene or operon requires cloning for each level to be tested. Using inducible promot