David K. Karig

Synthetic biology: new engineering rules for an emerging discipline

Synthetic biologists engineer complex artificial biological systems to investigate natural biological phenomena and for a variety of applications. We outline the basic features of synthetic biology as a new engineering discipline, covering examples from the latest literature and reflecting on the features that make it unique among all other existing engineering fields. We discuss methods for designing and constructing engineered cells with novel functions in a framework of an abstract hierarchy of biological devices, modules, cells, and multicellular systems. The classical engineering strategies of standardization, decoupling, and abstraction will have to be extended to take into account the inherent characteristics of biological devices and modules. To achieve predictability and reliability, strategies for engineering biology must include the notion of cellular context in the functional definition of devices and modules, use rational redesign and directed evolution for system optimization, and focus on accomplishing tasks using cell populations rather than individual cells. The discussion brings to light issues at the heart of designing complex living systems and provides a trajectory for future development.

Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium

Microbial consortia form when multiple species colocalize and communally generate a function that none is capable of alone. Consortia abound in nature, and their cooperative metabolic activities influence everything from biodiversity in the global food chain to human weight gain. Here, we present an engineered consortium in which the microbial members communicate with each other and exhibit a “consensus” gene expression response. Two colocalized populations of Escherichia coli converse bidirectionally by exchanging acyl-homoserine lactone signals. The consortium generates the gene-expression response if and only if both populations are present at sufficient cell densities. Because neither population can respond without the others signal, this consensus function can be considered a logical AND gate in which the inputs are cell populations. The microbial consensus consortium operates in diverse growth modes, including in a biofilm, where it sustains its response for several days.

Genetic circuit building blocks for cellular computation, communications, and signal processing

In this paper, we review an emerging engineering discipline to programcell behaviors by embedding synthetic gene networks that performcomputation, communications, and signal processing. To accomplishthis goal, we begin with a genetic component library and a biocircuitdesign methodology for assembling these components into compoundcircuits. The main challenge in biocircuit design lies in selectingwell-matched genetic components that when coupled, reliably producethe desired behavior. We use simulation tools to guide circuitdesign, a process that consists of selecting the appropriatecomponents and genetically modifying existing components until thedesired behavior is achieved. In addition to such rational design, wealso employ directed evolution to optimize genetic circuitbehavior. Building on Natures fundamental principle of evolution,this unique process directs cells to mutate their own DNA until theyfind gene network configurations that exhibit the desired systemcharacteristics. The integration of all the above capabilities infuture synthetic gene networks will enable cells to performsophisticated digital and analog computation, both asindividual entities and as part of larger cell communities. Thisengineering discipline and its associated tools will advance thecapabilities of genetic engineering, and allow us to harness cells fora myriad of applications not previously achievable.

Enlisting Hardware Architecture to Thwart Malicious Code Injection

Software vulnerabilities that enable the injection and execution of malicious code in pervasive Internet-connected computing devices pose serious threats to cyber security. In a common type of attack, a hostile party induces a software buffer overflow in a susceptible computing device in order to corrupt a procedure return address and transfer control to malicious code. These buffer overflow attacks are often employed to recruit oblivious hosts into distributed denial of service (DDoS) attack networks, which ultimately launch devastating DDoS attacks against victim networks or machines. In spite of existing software countermeasures that seek to prevent buffer overflow exploits, many systems remain vulnerable.

A processor architecture defense against buffer overflow attacks

Buffer overflow vulnerabilities in the memory stack continue to pose serious threats to network and computer security. By exploiting these vulnerabilities, a malicious party can strategically overwrite the return address of a procedure call, obtain control of a system, and subsequently launch more virulent attacks. Software countermeasures for such intrusions entail modifications to applications, compilers, and operating systems. Despite the availability of these defenses, many systems remain vulnerable to buffer overflow attacks. We present a hardware-based solution that prevents buffer overflow attacks involving procedure return address corruption. We add a secure return address stack to the processor that provides built-in, dynamic protection against return address tampering without requiring any effort by users or application programmers. Also, the performance impact is negligible for most applications. Changes are not required of application source code, so both legacy and future software can enjoy the security benefits of this solution.

Expression optimization and synthetic gene networks in cell-free systems

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

Noise in biological circuits

Noise biology focuses on the sources, processing, and biological consequences of the inherent stochastic fluctuations in molecular transitions or interactions that control cellular behavior. These fluctuations are especially pronounced in small systems where the magnitudes of the fluctuations approach or exceed the mean value of the molecular population. Noise biology is an essential component of nanomedicine where the communication of information is across a boundary that separates small synthetic and biological systems that are bound by their size to reside in environments of large fluctuations. Here we review the fundamentals of the computational, analytical, and experimental approaches to noise biology. We review results that show that the competition between the benefits of low noise and those of low population has resulted in the evolution of genetic system architectures that produce an uneven distribution of stochasticity across the molecular components of cells and, in some cases, use noise to drive biological function. We review the exact and approximate approaches to gene circuit noise analysis and simulation, and review many of the key experimental results obtained using flow cytometry and time-lapse fluorescent microscopy. In addition, we consider the probative value of noise with a discussion of using measured noise properties to elucidate the structure and function of the underlying gene circuit. We conclude with a discussion of the frontiers of and significant future challenges for noise biology.

Antibiotics as a selective driver for conjugation dynamics

It is generally assumed that antibiotics can promote horizontal gene transfer. However, because of a variety of confounding factors that complicate the interpretation of previous studies, the mechanisms by which antibiotics modulate horizontal gene transfer remain poorly understood. In particular, it is unclear whether antibiotics directly regulate the efficiency of horizontal gene transfer, serve as a selection force to modulate population dynamics after such gene transfer has occurred, or both. Here, we address this question by quantifying conjugation dynamics in the presence and absence of antibiotic-mediated selection. Surprisingly, we find that sublethal concentrations of antibiotics from the most widely used classes do not significantly increase the conjugation efficiency. Instead, our modelling and experimental results demonstrate that conjugation dynamics are dictated by antibiotic-mediated selection, which can both promote and suppress conjugation dynamics. Our findings suggest that the contribution of antibiotics to the promotion of horizontal gene transfer may have been overestimated. These findings have implications for designing effective antibiotic treatment protocols and for assessing the risks of antibiotic use.

Probing Cell-Free Gene Expression Noise in Femtoliter Volumes

Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell-relevant 20 fL volumes (between the volumes of Escherichia coli and Saccharomyces cerevisiae ), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely among different containers, likely due to non-Poisson distribution of expression machinery at the observed scale. Previously, this phenomenon has been observed only in liposomes. In addition, we analyze gene expression noise. This analysis is facilitated by our use of cell-free systems, which allow the mapping of the measured noise properties to intrinsic noise models. In contrast, previous live cell noise analysis efforts have been complicated by multiple noise sources. Noise analysis reveals signatures of translational bursting, while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

Multi-Input Regulation and Logic with T7 Promoters in Cells and Cell-Free Systems

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.