Timothy K. Lu

Dispersing biofilms with engineered enzymatic bacteriophage

Synthetic biology involves the engineering of biological organisms by using modular and generalizable designs with the ultimate goal of developing useful solutions to real-world problems. One such problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by host immune systems. To address this issue, we engineered bacteriophage to express a biofilm-degrading enzyme during infection to simultaneously attack the bacterial cells in the biofilm and the biofilm matrix, which is composed of extracellular polymeric substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy is significantly greater than that of nonenzymatic bacteriophage treatment. Our engineered enzymatic phage substantially reduced bacterial biofilm cell counts by ≈4.5 orders of magnitude (≈99.997% removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic biology to an important medical and industrial problem.

Synthetic Gene Networks That Count

One, Two, Three Synthetic biologists take much of their inspiration from computing and electrical engineering, attempting to gain programmability in cells using analogous genetic circuits. Friedland et al. (p. 1199; see the Perspective by Smolke) have constructed complementary Escherichia coli synthetic gene networks that keep track of prior molecular events to function as counters. These modular devices count up to three induction events over a range of frequencies and could be extended to higher numbers. Similar devices may find application in multi-event processes of cell biology, bioengineering, and, potentially, therapeutics. Genetic counters with the ability to count up to three induction events are constructed in Escherichia coli. Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.

Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy

Antimicrobial drug development is increasingly lagging behind the evolution of antibiotic resistance, and as a result, there is a pressing need for new antibacterial therapies that can be readily designed and implemented. In this work, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. We show that suppressing the SOS network in Escherichia coli with engineered bacteriophage enhances killing by quinolones by several orders of magnitude in vitro and significantly increases survival of infected mice in vivo. In addition, we demonstrate that engineered bacteriophage can enhance the killing of antibiotic-resistant bacteria, persister cells, and biofilm cells, reduce the number of antibiotic-resistant bacteria that arise from an antibiotic-treated population, and act as a strong adjuvant for other bactericidal antibiotics (e.g., aminoglycosides and β-lactams). Furthermore, we show that engineering bacteriophage to target non-SOS gene networks and to overexpress multiple factors also can produce effective antibiotic adjuvants. This work establishes a synthetic biology platform for the rapid translation and integration of identified targets into effective antibiotic adjuvants.

Next-generation synthetic gene networks

Synthetic biology is focused on the rational construction of biological systems based on engineering principles. During the fields first decade of development, significant progress has been made in designing biological parts and assembling them into genetic circuits to achieve basic functionalities. These circuits have been used to construct proof-of-principle systems with promising results in industrial and medical applications. However, advances in synthetic biology have been limited by a lack of interoperable parts, techniques for dynamically probing biological systems and frameworks for the reliable construction and operation of complex, higher-order networks. As these challenges are addressed, synthetic biologists will be able to construct useful next-generation synthetic gene networks with real-world applications in medicine, biotechnology, bioremediation and bioenergy.

Synthetic circuits integrating logic and memory in living cells

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications.

Synthetic analog computation in living cells

A central goal of synthetic biology is to achieve multi-signal integration and processing in living cells for diagnostic, therapeutic and biotechnology applications. Digital logic has been used to build small-scale circuits, but other frameworks may be needed for efficient computation in the resource-limited environments of cells. Here we demonstrate that synthetic analog gene circuits can be engineered to execute sophisticated computational functions in living cells using just three transcription factors. Such synthetic analog gene circuits exploit feedback to implement logarithmically linear sensing, addition, ratiometric and power-law computations. The circuits exhibit Weber’s law behaviour as in natural biological systems, operate over a wide dynamic range of up to four orders of magnitude and can be designed to have tunable transfer functions. Our circuits can be composed to implement higher-order functions that are well described by both intricate biochemical models and simple mathematical functions. By exploiting analog building-block functions that are already naturally present in cells, this approach efficiently implements arithmetic operations and complex functions in the logarithmic domain. Such circuits may lead to new applications for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosensing or would benefit from the fine control of gene expression.

Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells

RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.

A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.

Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas.

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function. Here, we show that the CRISPR/Cas system of Streptococcus pyogenes can be programmed to direct both activation and repression to natural and artificial eukaryotic promoters through the simple engineering of guide RNAs with base-pairing complementarity to target DNA sites. We demonstrate that the activity of CRISPR-based transcription factors (crisprTFs) can be tuned by directing multiple crisprTFs to different positions in natural promoters and by arraying multiple crisprTF-binding sites in the context of synthetic promoters in yeast and human cells. Furthermore, externally controllable regulatory modules can be engineered by layering gRNAs with small molecule-responsive proteins. Additionally, single nucleotide substitutions within promoters are sufficient to render them orthogonal with respect to the same gRNA-guided crisprTF. We envision that CRISPR-based eukaryotic gene regulation will enable the facile construction of scalable synthetic gene circuits and open up new approaches for mapping natural gene networks and their effects on complex cellular phenotypes.

The next generation of bacteriophage therapy

Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.