Engineering intelligent chassis cells via recombinase-based MEMORY circuits.

Synthetic biologists seek to engineer intelligent living systems capable of decision-making, communication, and memory. Separate technologies exist for each tenet of intelligence; however, the unification of all three properties in a living system has not been achieved. Here, we engineer completely intelligent Escherichia coli strains that harbor six orthogonal and inducible genome-integrated recombinases, forming Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY). MEMORY chassis cells facilitate intelligence via the discrete multi-input regulation of recombinase functions enabling inheritable DNA inversions, deletions, and genomic insertions. MEMORY cells can achieve programmable and permanent gain (or loss) of functions extrachromosomally or from a specific genomic locus, without the loss or modification of the MEMORY platform - enabling the sequential programming and reprogramming of DNA circuits within the cell. We demonstrate all three tenets of intelligence via a probiotic (Nissle 1917) MEMORY strain capable of information exchange with the gastrointestinal commensal Bacteroides thetaiotaomicron.

Engineering allosteric transcription factors guided by the LacI topology.

Allosteric transcription factors (aTFs) are used in a myriad of processes throughout biology and biotechnology. aTFs have served as the workhorses for developments in synthetic biology, fundamental research, and protein manufacturing. One of the most utilized TFs is the lactose repressor (LacI). In addition to being an exceptional tool for gene regulation, LacI has also served as an outstanding model system for understanding allosteric communication. In this perspective, we will use the LacI TF as the principal exemplar for engineering alternate functions related to allostery-i.e., alternate protein DNA interactions, alternate protein-ligand interactions, and alternate phenotypic mechanisms. In addition, we will summarize the design rules and heuristics for each design goal and demonstrate how the resulting design rules and heuristics can be extrapolated to engineer other aTFs with a similar topology-i.e., from the broader LacI/GalR family of TFs.

Next generation synthetic memory via intercepting recombinase function.

Here we present a technology to facilitate synthetic memory in a living system via repurposing Transcriptional Programming (i.e., our decision-making technology) parts, to regulate (intercept) recombinase function post-translation. We show that interception synthetic memory can facilitate programmable loss-of-function via site-specific deletion, programmable gain-of-function by way of site-specific inversion, and synthetic memory operations with nested Boolean logical operations. We can expand interception synthetic memory capacity more than 5-fold for a single recombinase, with reconfiguration specificity for multiple sites in parallel. Interception synthetic memory is ~10-times faster than previous generations of recombinase-based memory. We posit that the faster recombination speed of our next-generation memory technology is due to the post-translational regulation of recombinase function. This iteration of synthetic memory is complementary to decision-making via Transcriptional Programming - thus can be used to develop intelligent synthetic biological systems for myriad applications.

Performance Prediction of Fundamental Transcriptional Programs.

Transcriptional programming leverages systems of engineered transcription factors to impart decision-making (e.g., Boolean logic) in chassis cells. The number of components used to construct said decision-making systems is rapidly increasing, making an exhaustive experimental evaluation of iterations of biological circuits impractical. Accordingly, we posited that a predictive tool is needed to guide and accelerate the design of transcriptional programs. The work described here involves the development and experimental characterization of a large collection of network-capable single-INPUT logical operations─i.e., engineered BUFFER (repressor) and engineered NOT (antirepressor) logical operations. Using this single-INPUT data and developed metrology, we were able to model and predict the performances of all fundamental two-INPUT compressed logical operations (i.e., compressed AND gates and compressed NOR gates). In addition, we were able to model and predict the performance of compressed mixed phenotype logical operations (A NIMPLY B gates and complementary B NIMPLY A gates). These results demonstrate that single-INPUT data is sufficient to accurately predict both the qualitative and quantitative performance of a complex circuit. Accordingly, this work has set the stage for the predictive design of transcriptional programs of greater complexity.

Transcriptional programming in a Bacteroides consortium.

Bacteroides species are prominent members of the human gut microbiota. The prevalence and stability of Bacteroides in humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community of Bacteroides (consortium transcriptional programming) with genetic circuit compression. Circuit compression requires systematic pairing of engineered transcription factors with cognate regulatable promoters. In turn, we demonstrate the compression workflow by designing, building, and testing all fundamental two-input logic gates dependent on the inputs isopropyl-ß-D-1-thiogalactopyranoside and D-ribose. We then deploy complete sets of logical operations in five human donor Bacteroides, with which we demonstrate sequential gain-of-function control in co-culture. Finally, we couple transcriptional programs with CRISPR interference to achieve loss-of-function regulation of endogenous genes-demonstrating complex control over community composition in co-culture. This work provides a powerful toolkit to program gene expression in Bacteroides for the development of bespoke therapeutic bacteria.

Biological signal processing filters via engineering allosteric transcription factors.

Signal processing is critical to a myriad of biological phenomena (natural and engineered) that involve gene regulation. Biological signal processing can be achieved by way of allosteric transcription factors. In canonical regulatory systems (e.g., the lactose repressor), an INPUT signal results in the induction of a given transcription factor and objectively switches gene expression from an OFF state to an ON state. In such biological systems, to revert the gene expression back to the OFF state requires the aggressive dilution of the input signal, which can take 1 or more d to achieve in a typical biotic system. In this study, we present a class of engineered allosteric transcription factors capable of processing two-signal INPUTS, such that a sequence of INPUTS can rapidly transition gene expression between alternating OFF and ON states. Here, we present two fundamental biological signal processing filters, BANDPASS and BANDSTOP, that are regulated by D-fucose and isopropyl-ß-D-1-thiogalactopyranoside. BANDPASS signal processing filters facilitate OFF-ON-OFF gene regulation. Whereas, BANDSTOP filters facilitate the antithetical gene regulation, ON-OFF-ON. Engineered signal processing filters can be directed to seven orthogonal promoters via adaptive modular DNA binding design. This collection of signal processing filters can be used in collaboration with our established transcriptional programming structure. Kinetic studies show that our collection of signal processing filters can switch between states of gene expression within a few minutes with minimal metabolic burden-representing a paradigm shift in general gene regulation.

Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors.

Recent advances in synthetic biology and protein engineering have increased the number of allosteric transcription factors used to regulate independent promoters. These developments represent an important increase in our biological computing capacity, which will enable us to construct more sophisticated genetic programs for a broad range of biological technologies. However, the majority of these transcription factors are represented by the repressor phenotype (BUFFER), and require layered inversion to confer the antithetical logical function (NOT), requiring additional biological resources. Moreover, these engineered transcription factors typically utilize native ligand binding functions paired with alternate DNA binding functions. In this study, we have advanced the state-of-the-art by engineering and redesigning the PurR topology (a native antirepressor) to be responsive to caffeine, while mitigating responsiveness to the native ligand hypoxanthine-i.e., a deamination product of the input molecule adenine. Importantly, the resulting caffeine responsive transcription factors are not antagonized by the native ligand hypoxanthine. In addition, we conferred alternate DNA binding to the caffeine antirepressors, and to the PurR scaffold, creating 38 new transcription factors that are congruent with our current transcriptional programming structure. Finally, we leveraged this system of transcription factors to create integrated NOR logic and related feedback operations. This study represents the first example of a system of transcription factors (antirepressors) in which both the ligand binding site and the DNA binding functions were successfully engineered in tandem.

Biomolecular Systems Engineering: Unlocking the Potential of Engineered Allostery via the Lactose Repressor Topology.

Allosteric function is a critical component of many of the parts used to construct gene networks throughout synthetic biology. In this review, we discuss an emerging field of research and education, biomolecular systems engineering, that expands on the synthetic biology edifice-integrating workflows and strategies from protein engineering, chemical engineering, electrical engineering, and computer science principles. We focus on the role of engineered allosteric communication as it relates to transcriptional gene regulators-i.e., transcription factors and corresponding unit operations. In this review, we (a) explore allosteric communication in the lactose repressor LacI topology, (b) demonstrate how to leverage this understanding of allostery in the LacI system to engineer non-natural BUFFER and NOT logical operations, (c) illustrate how engineering workflows can be used to confer alternate allosteric functions in disparate systems that share the LacI topology, and (d) demonstrate how fundamental unit operations can be directed to form combinational logical operations.

Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget.

Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden - as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.

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

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 ß-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.

Pathways and Challenges for Biomimetic Desalination Membranes with Sub-Nanometer Channels.

Transmembrane protein channels, including ion channels and aquaporins that are responsible for fast and selective transport of water, have inspired membrane scientists to exploit and mimic their performance in membrane technologies. These biomimetic membranes comprise discrete nanochannels aligned within amphiphilic matrices on a robust support. While biological components have been used directly, extensive work has also been conducted to produce stable synthetic mimics of protein channels and lipid bilayers. However, the experimental performance of biomimetic membranes remains far below that of biological membranes. In this review, we critically assess the status and potential of biomimetic desalination membranes. We first review channel chemistries and their transport behavior, identifying key characteristics to optimize water permeability and salt rejection. We compare various channel types within an industrial context, considering transport performance, processability, and stability. Through a re-examination of previous vesicular stopped-flow studies, we demonstrate that incorrect permeability equations result in an overestimation of the water permeability of nanochannels. We find in particular that the most optimized aquaporin-bearing bilayer had a pure water permeability of 2.1 L m-2 h-1 bar-1, which is comparable to that of current state-of-the-art polymeric desalination membranes. Through a quantitative assessment of biomimetic membrane formats, we analytically show that formats incorporating intact vesicles offer minimal benefit, whereas planar biomimetic selective layers could allow for dramatically improved salt rejections. We then show that the persistence of nanoscale defects explains observed subpar performance. We conclude with a discussion on optimal strategies for minimizing these defects, which could enable breakthrough performance.

Engineering allosteric communication.

Protein allostery is a vitally important protein function that has proven to be a vexing problem to understand at the molecular level. Allosteric communication is a hallmark of many protein functions. However, despite more than four decades of study the details regarding allosteric communication in protein systems are still being developed. Engineering of LacI and related homologues to confer alternate allosteric communication has shed light on the pre-requisites for the de novo design of allosteric communication. While the de novo design of an allosteric pathway and complementary functional surfaces has not been realized, this review highlights recent advances that set the stage for true predictive design for a given protein topology.

Transcriptional programming using engineered systems of transcription factors and genetic architectures.

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.

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

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 ß-d-1-thiogalactopyranoside (IPTG). Recently, laboratory evolution was used to confer inverted function in the native LacI topology resulting in anti-LacI (antilac) function (IAYQR), where IPTG binding results in gene suppression. Here we engineered 46 antilacs with alternate DNA binding function (IAADR). Phenotypically, IAADR transcription factors are the inverse of wild-type I+YQR function and possess alternate DNA recognition (ADR). This collection of bespoke IAADR bind orthogonally to disparate non-natural operator DNA sequences and suppress gene expression in the presence of IPTG. This new class of IAADR 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 IAADR identified in this study are also orthogonal to the naturally occurring operator O1. 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 IAADR 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).

Biomolecular Assemblies: Moving from Observation to Predictive Design.

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.

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

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sensing is the conversion of an exogenous signal, usually a small molecule or environmental cue such as a protein-ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure-function relationship of LacI; (2) discuss how the physical attributes of LacI can be leveraged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI. WIREs Nanomed Nanobiotechnol 2017, 9:e1461. doi: 10.1002/wnan.1461 For further resources related to this article, please visit the WIREs website.

Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions.

The lactose repressor (LacI) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of LacI for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the LacI scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the LacI topology that are responsive to isopropyl ß-d-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media. The new variants exhibit repressive as well as antilac (i.e., inverse-repression + IPTG) functions and variations in the control of gene output upon exposure to different concentrations of IPTG. In addition, examination of this collection of variants in solution results in the controlled output of a canonical florescent reporter, demonstrating the utility of this collection of new regulatory proteins under standard conditions.

Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation.

Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.