In vivo and in silico approaches to study the effects of copy number variation on gene networks behaviour.View all posters
University of Pavia, Italy
One of the main problems connected with the assembly of genetic circuits from simple genetic parts is the unpredictability of the behaviour of the whole system, given the input/output behaviour of the single modules that compose it. Within this frame, it is essential to trace the linearity working boundaries of engineered biological systems to rationally design gene networks. In this study, the nonlinear and saturation effects due to copy number variation were examined using in vivo and in silico methodologies. First, a BioBrick-compatible integrative vector for Escherichia coli was designed, constructed and validated. This tool supports both site specific and homologous recombination and has been successfully used to target two different loci of the E. coli chromosome. This tool allows the construction of gene networks present in a single copy in the bacterial genome. Commonly used plasmids present in the Registry were also used. They enable a 40-fold copy number variation (from 5 to about 200 copies per cell). Quantitative analysis of the activity of different constitutive promoters was measured in the genomic and plasmid contexts. Secondly, a simple inducible device in four different copy number contexts (from 1 to about 200 copies per cell) was quantitatively characterized. An empiric mathematical model was used to fully characterize the device in all the investigated conditions. Finally, a mechanistic model of the inducible device, based on the mass action law, has been proposed and used to study the effects of copy number variation of both promoter and transcription factor, individually and in concert. Taken together, these results show how fundamental the copy number tuning is in regulating gene networks. Biological and computational tools to study these phenomena have been developed and tested and an important step has been done in the process of defining the linear working boundaries of synthetic biological devices.