Transcriptional programming using engineered systems of transcription factors and genetic architectures

Abstract

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, and protein manufacturing. The most successful approaches to date are based on modulating mRNA synthesis via an inducible coupling to transcriptional effectors. Here we present a biological programming structure that leverages a system of engineered transcription factors and complementary genetic architectures. We use a modular design strategy to create 27 non-natural and non-synonymous transcription factors using the lactose repressor topology as a guide. To direct systems of engineered transcription factors we employ parallel and series genetic (DNA) architectures and confer fundamental and combinatorial logical control over gene expression. Here we achieve AND, OR, NOT, and NOR logical controls in addition to two non-canonical half-AND operations. The basic logical operations and corresponding parallel and series genetic architectures represent the building blocks for subsequent combinatorial programs, which display both digital and analog performance.

Engineered systems of inducible anti-repressors for the next generation of biological programming

“…5). To accomplish this, we employed a modular design strategy that we developed in a previous study 25 . Here, the base design space was given by 3 distinct E-RCD and 6 ADR modules; however, each E-RCD had two or more allosteric solutions (Fig.…”

“…In principle, BUO can be developed and defined to a similar level of objective performance-provided we have a metrology to define the performance boundary. In our previous study, we established a metrology for 35 (repressor X + ADR based) BUFFER unit operations 25 . In brief, this metrology consists of three parts: (i) defining the conditional ).…”

“…3e). We used series and parallel in our previous report to construct a broad range of repressor based BUFFER combinatorial operations and related biological programs 25 . Series-parallel was tangentially introduced in a previous report by Shis et al 43 , and here we formalized this architecture as part of a larger transcriptional programming edifice.…”

“…In principle, this is akin to engineering a computer program to carry out a desired task. Recent advances in synthetic biology have brought the fields of biomolecular, electrical, chemical, and computer engineering far closer [4][5][6] , for instance with the development of biological sensors [7][8][9] , memory [10][11][12] , switches and controllers [13][14][15][16][17] , transistors 18 , counters 19 , oscillators and clocks [20][21][22][23][24] , and equivalents of Boolean logic gates [25][26][27] . In many of these developments, transcriptional regulation is achieved via allosteric transcription factors (TFs) 28 .…”

“…A similar modular design approach was used recently to engineer a collection of TFs not observed in nature with a range of DNA and ligand binding affinities. These TFs were then deployed in gene networks that developed the basic logical operations AND 25,43 , OR, NOT (inverted) 26,38 , and NOR (apparent) 25 , along with other combinatorial logical operations 25 . In addition, Chan et al have used an analogous modular design strategy to engineer TF repressors for other biotechnological applications (e.g., Deadman and passcode kill switches) 38 .…”