Deconstruction of complex protein signaling switches: a roadmap toward engineering higher‐order gene regulators

Davey, James A.; Wilson, Clive

doi:10.1002/wnan.1461

Cited by 6 publications

(7 citation statements)

References 39 publications

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“…Repressors function by binding to a promoter and physically compromising binding by RNAP until acted upon by an exogenous signal 31 . In recent years, efforts have sought to increase cellular computing capacity and complexity through the development of additional tools for transcriptional regulation 32 . These TFs, though, have been widely confined to repressors functional with only a small number of operator (DNA) sequences, limiting their application in biological circuit engineering [33][34][35][36][37] .…”

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confidence: 99%

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

et al. 2020

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Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a workflow for engineering systems of non-natural anti-repressors. In this study, we create 41 inducible anti-repressors. This collection of transcription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl β-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series-parallel genetic architectures represent a nascent anti-repressor based transcriptional programming structure.

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confidence: 99%

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

et al. 2020

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“…X were selected (opposed to all 14 published I A X 13 ) based on performance metrics in solution. Using the modular protein design strategy we proposed in Davey and Wilson, 17 54 putative antilacs with alternate DNA recognition (I A ADR ) were constructed. In turn, we assessed the performance metrics for all 54 I A ADR (plus nine wild-type controls I A YQR ), using the single operator system we engineered for this study.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Engineering a New Class of Anti-LacI Transcription Factors with Alternate DNA Recognition

Rondon

Wilson

2019

ACS Synth. Biol.

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The lactose repressor, LacI (I + YQR ), is an archetypal transcription factor that has been a workhorse in many synthetic genetic networks. LacI represses gene expression (apo ligand) and is induced upon binding of the ligand isopropyl β-D-1-thiogalactopyranoside (IPTG). Recently, laboratory evolution was used to confer inverted function in the native LacI topology resulting in anti-LacI (antilac) function (I A YQR ), where IPTG binding results in gene suppression. Here we engineered 46 antilacs with alternate DNA binding function (I A ADR ). Phenotypically, I A ADR transcription factors are the inverse of wild-type I + YQR function and possess alternate DNA recognition (ADR). This collection of bespoke I A ADR bind orthogonally to disparate nonnatural operator DNA sequences and suppress gene expression in the presence of IPTG. This new class of I A ADR gene regulators were designed modularly via the systematic pairing of nine alternate allosteric regulatory cores with six alternate DNA binding domains that interact with complementary synthetic operator DNA sequences. The 46 I A ADR identified in this study are also orthogonal to the naturally occurring operator O 1 . Finally, a demonstration of full orthogonality was achieved via the construction of synthetic genetic toggle switches composed of two nonsynonymous unit pair operations that control two distinct fluorescent outputs. This new class of I A ADR transcription factors will facilitate the expansion of the computational capacity of engineered gene circuits, via the scalable increase in the control over the number of gene outputs by way of the expansion of the number of unique transcription factors (or systems of transcription factors) that can simultaneously regulate one or more promoter(s).

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“…358,359 The lactose repressor (LacI) is the standard model system for studying allostery and understanding how transcription is controlled by using small metabolites to modulate protein association with specific DNA sites. 360 In a recent study, Wilson et al used random mutagenesis laboratory evolution to re-route allosteric communication in the LacI scaffold to confer anti-lac functionality. 361,362 (Figure 19).…”

Section: Transcription Factor Assemblymentioning

confidence: 99%

Biomolecular Assemblies: Moving from Observation to Predictive Design

et al. 2018

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Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.

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Deconstruction of complex protein signaling switches: a roadmap toward engineering higher‐order gene regulators

Cited by 6 publications

References 39 publications

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

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

Engineering a New Class of Anti-LacI Transcription Factors with Alternate DNA Recognition

Biomolecular Assemblies: Moving from Observation to Predictive Design

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