Synthetic Biology Group, Professor Timothy Lu

The Synthetic Biology Group is focused on advancing fundamental designs and applications for synthetic biology. Using principles inspired by electrical engineering and computer science, we are developing new techniques for constructing, probing, modulating, and modeling engineered biological circuits.  Our current application areas include infectious diseases, amyloid-associated conditions, and nanotechnology.

Living Functional Materials.

Many natural biological systems—such as biofilms, shells and skeletal tissues—are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. By using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells. [Nature Materials 2014]

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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 applications1. 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 applica- tions for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosen- sing or would benefit from the fine control of gene expression. [Nature 2013]

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Integrated Logic and Memory in Living Cells.

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. To integrate these into individual living cells, we have created 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 enables the creation of arbitrary Boolean logic functions in living cells without requiring cascades comprising multiple logic gates. Moreover, this platform enables long-term maintenance of memory (for example, we have validated stability 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 multi-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. [Nature Biotechnology 2013]

Synthetic circuits integrating logic and memory in living cells

[Image Credit: Liang Zong and Yan Liang]

Scalable Toolkits for Engineering Transcriptional Regulation in Eukaryotes.

Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. We have engineered a scalable 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 under- standing the design principles of eukaryotic transcriptional complexes and networks. [Cell 2012]

synthetic transcription factors from cell 2012

Engineered Bacteriophage Therapeutics for Antibiotic-Resistant Infections.

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. [PNAS 2007]

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. To tackle this problem, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. We showed 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 beta-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. [PNAS 2009]

To place our work into context, 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. [Current Opinion in Microbiology 2011]

engineered phage from curr opin micro 2011