Programmable T7-based synthetic transcription factors.

Abstract

Despite recent progress on synthetic transcription factor generation in eukaryotes, there remains a need for high-activity bacterial versions of these systems. In synthetic biology applications, it is useful for transcription factors to have two key features: they should be orthogonal (influencing only their own targets, with minimal off-target effects), and programmable (able to be directed to a wide range of user-specified transcriptional start sites). The RNA polymerase of the bacteriophage T7 has a number of appealing properties for synthetic biological designs: it can produce high transcription rates; it is a compact, single-subunit polymerase that has been functionally expressed in a variety of organisms; and its viral origin reduces the connection between its activity and that of its host's transcriptional machinery. We have created a system where a T7 RNA polymerase is recruited to transcriptional start sites by DNA binding proteins, either directly or bridged through protein–protein interactions, yielding a modular and programmable system for strong transcriptional activation of multiple orthogonal synthetic transcription factor variants in Escherichia coli. To our knowledge this is the first exogenous, programmable activator system in bacteria.