A Genetic Toggle for Chemical Control of Individual Plk1 Substrates.

Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1S (L197F) and Plk1T (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1S but not Plk1T sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.

Using Engineered Bacteria to Characterize Infection Dynamics and Antibiotic Effects In Vivo.

Synthetic biology has focused on engineering microbes to synthesize useful products or to serve as living diagnostics and therapeutics. Here we utilize a host-derived Escherichia coli strain engineered with a genetic toggle switch as a research tool to examine in vivo replicative states in a mouse model of chronic infection, and to compare in vivo and in vitro bacterial behavior. In contrast to the effect of antibiotics in vitro, we find that the fraction of actively dividing bacteria remains relatively high throughout the course of a chronic infection in vivo and increases in response to antibiotics. Moreover, the presence of non-dividing bacteria in vivo does not necessarily lead to an antibiotic-tolerant infection, in contrast to expectations from in vitro experiments. These results demonstrate the utility of engineered bacteria for querying pathogen behavior in vivo, and the importance of validating in vitro studies of antibiotic effects with in vivo models.

A Glucose-Sensing Toggle Switch for Autonomous, High Productivity Genetic Control.

Many biosynthetic strategies are coupled to growth, which is inherently limiting, as (1) excess feedstock (e.g., sugar) may be converted to biomass, instead of product, (2) essential genes must be maintained, and (3) growth toxicity must be managed. A decoupled growth and production phase strategy could avoid these issues. We have developed a toggle switch that uses glucose sensing to enable this two-phase strategy. Temporary glucose starvation precisely and autonomously activates product pathway expression in rich or minimal media, obviating the requirement for expensive inducers. The switch remains stably in the new state even after reintroduction of glucose. In the context of polyhydroxybutyrate (PHB) biosynthesis, our system enables shorter growth phases and comparable titers to a constitutively expressing PHB strain. This two-phase production strategy, and specifically the glucose toggle switch, should be broadly useful to initiate many types of genetic program for metabolic engineering applications.

Towards programmable plant genetic circuits.

Synthetic biology enables the construction of genetic circuits with predictable gene functions in plants. Detailed quantitative descriptions of the transfer function or input-output function for genetic parts (promoters, 5' and 3' untranslated regions, etc.) are collected. These data are then used in computational simulations to determine their robustness and desired properties, thereby enabling the best components to be selected for experimental testing in plants. In addition, the process forms an iterative workflow which allows vast improvement to validated elements with sub-optimal function. These processes enable computational functions such as digital logic in living plants and follow the pathway of technological advances which took us from vacuum tubes to cell phones.

Hypothetical biomolecular probe based on a genetic switch with tunable symmetry and stability.

BACKGROUND: Genetic switches are ubiquitous in nature, frequently associated with the control of cellular functions and developmental programs. In the realm of synthetic biology, it is of great interest to engineer genetic circuits that can change their mode of operation from monostable to bistable, or even to multistable, based on the experimental fine-tuning of readily accessible parameters. In order to successfully design robust, bistable synthetic circuits to be used as biomolecular probes, or understand modes of operation of such naturally occurring circuits, we must identify parameters that are key in determining their characteristics. RESULTS: Here, we analyze the bistability properties of a general, asymmetric genetic toggle switch based on a chemical-reaction kinetic description. By making appropriate approximations, we are able to reduce the system to two coupled differential equations. Their deterministic stability analysis and stochastic numerical simulations are in excellent agreement. Drawing upon this general framework, we develop a model of an experimentally realized asymmetric bistable genetic switch based on the LacI and TetR repressors. By varying the concentrations of two synthetic inducers, doxycycline and isopropyl ß-D-1-thiogalactopyranoside, we predict that it will be possible to repeatedly fine-tune the mode of operation of this genetic switch from monostable to bistable, as well as the switching rates over many orders of magnitude, in an experimental setting. Furthermore, we find that the shape and size of the bistability region is closely connected with plasmid copy number. CONCLUSIONS: Based on our numerical calculations of the LacI-TetR asymmetric bistable switch phase diagram, we propose a generic work-flow for developing and applying biomolecular probes: Their initial state of operation should be specified by controlling inducer concentrations, and dilution due to cellular division would turn the probes into memory devices in which information could be preserved over multiple generations. Additionally, insights from our analysis of the LacI-TetR system suggest that this particular system is readily available to be employed in this kind of probe.

Development of a stress-induced mutagenesis module for autonomous adaptive evolution of Escherichia coli to improve its stress tolerance.

BACKGROUND: Microbial tolerance to different environmental stresses is of importance for efficient production of biofuels and biochemical. Such traits are often improved by evolutionary engineering approaches including mutagen-induced mutagenesis and successive passage. In contrast to these approaches which generate mutations in rapidly growing cells, recent research showed that mutations could be generated in non-dividing cells under stressful but non-lethal conditions, leading to the birth of the theory of stress-induced mutagenesis (SIM). A molecular mechanism of SIM has been elucidated to be mutagenic repair of DNA breaks. This inspired us to develop a synthetic SIM module to simulate the mutagenic cellular response so as to accelerate microbial adaptive evolution for an improved stress tolerance. RESULTS: A controllable SIM evolution module was devised based on a genetic toggle switch in Escherichia coli. The synthetic module enables expression and repression of the genes related to up- and down-regulation responses during SIM in a bistable way. Upon addition of different inducers, the module can be turned on or off, triggering transition to a mutagenic or a high-fidelity state and thus allowing periodic adaptive evolution. Six genes (recA, dinB, umuD, ropS, ropE, and nusA) in the up-regulation responses were evaluated for their potentials to enhance the SIM rate. Expression of recA, dinB, or ropS alone increased the SIM rate by 4.5- to 13.7-fold, whereas their combined expression improved the rate by 31.9-fold. Besides, deletion of mutL increased the SIM rate by 82-fold. Assembly of these genes into the SIM module in the mutL-deletion E. coli strain elevated the SIM rate by nearly 3000-fold. Accelerated adaptive evolution of E. coli equipped with this synthetic SIM module was demonstrated under n-butanol stress, with the minimal inhibitory concentration of n-butanol increasing by 56 % within 2.5 months. CONCLUSIONS: A synthetic SIM module was constructed to simulate cellular mutagenic responses during SIM. Based on this, a novel evolutionary engineering approach-SIM-based adaptive evolution-was developed to improve the n-butanol tolerance of E. coli.

Noise-induced Synchronized Switching of a Multicellular System / 生物化学与生物物理进展

Genetic bistable systems are a large class of important biological systems. Bistability, the capacity to achieve two distinct stable steady states in response to a set of external stimuli, arises within biological systems ranging from the ? phage switch in bacteria to cellular signal transduction pathways in mammalian cells. On the other hand, the increasing experimental evidence in the form of bimodal population distribution has indicated that noise plays a very key role in the switching of bistable systems. However, the physiological mechanism underling noise-induced switching behaviors has not been well explored yet. In the previous work, it has been showed that noise can induce coherent switch for a single genetic Toggle switch system. Here the influence of several kinds of noises (including intracellular and extracellular noises) on synchronized switch was investigated for a multicell gene toggle switch network system. It has been found that multiplicative noises resulting from fluctuations of either synthesis or degradation rates and the additive noise within each cell (they altogether are called as intracellular noises) all can induce the synchronized switch, and that there exists an optimal noise intensity such that the synchronized switch is optimally achieved and the amplification factor has the maximal value. On the other hand, the extracellular noises arising from the stochastic fluctuation of the cellular environment, not only brings about the synchronized switch, but also enhances it by suppressing intracellular fluctuations when the intracellular noises are not enough to induce the synchronized switch. Finally, the influence of the diffusive rate of signal molecules affected by noise on the dynamics of the multicellular system was also investigated, showing that the larger the diffusive rate, the better the synchronized switch and the larger the amplification factor.