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.

Guiding biomolecular interactions in cells using de novo protein-protein interfaces

“…We are working on this challenge using the ISAMBARD suite for computational protein design. 44,45 Nonetheless, our results, together with those of others, 26,28 indicate that the rules used to design our peptide sequences are sufficiently comprehensive to allow the CC components of the sequences to interact as designed in a cellular environment. Although we have yet to probe these systems with proteomics, it appears that the introduced biomolecular interactions operate orthogonally to the endogenous E. coli proteome and interactome.…”

“…Some of the peptide sequences tested here have been shown recently to assemble in E. coli in other contexts: the heterodimeric CC drives the assembly of a novel cytoscaffold and the subcellular localization of active enzymes when fused to shell proteins of a bacterial microcompartment; 6,7 and, while the work presented here was in preparation, the same heterodimeric CCs have been shown by others to recruit T7 RNA polymerase to Zn-finger DNA-binding domains. 28 Some adverse context-dependent effects have been noted: in our activation experiments proximity effects may inhibit heterodimerization; and in the programmable T7 RNA polymerase system the hierarchy of CC interaction strength varies with the nature of the Zn-finger domains to which the peptides are fused. 28 Thus, it is likely that improved rules or methods for designing linker sequences will be needed to help minimize such effects in future applications.…”

“…Natural and synthetic CCs have been shown to function effectively as PPIs within transcription activators in yeast and E. coli. 25,28 Here, we test the ability of the de novo designed homo and hetero-dimeric CC peptides to function as PPI domains in a range of contexts within living E. coli cells. We find that the CC peptides can mediate PPIs in multiple systems in vivo , including as part of a cytoplasmic split enzyme, and as components of both transcription repressors and transcription activators.…”

Evolution of C‐Terminal Modification Tolerance in Full‐Length and Split T7 RNA Polymerase Biosensors

“…Moreover, the evolved split RNAP system can still be used to easily generate biosensors when coupled to our previously evolved proximity‐dependent split RNAP biosensor platform. Other T7 RNAP‐based engineered systems currently in development will likely also benefit from this expanded engineering flexibility …”

Combining rational design and continuous evolution on minimalist proteins that target DNA

“…Thus, the dimerization interface comprising the E47 HLH and FosW LZ should only homodimerize and not fortuitously partner with an endogenous protein in any type of cell. This feature of protein-partner exclusivity could be useful in in vivo applications, such as synthetic biology where a designed TF can work exclusively on an engineered target within a cell, thereby minimizing off-target effects, 62 and in development of protein-based drugs that target a specific disease, without interactions with endogenous proteins that led to unwanted effects. 7 We used rational design to generate mutants with the FosW LZ appended to Helix 2 of the ME47/AA bHLH domain.…”