A geometric and structural approach to the analysis and design of biological circuit dynamics: a theory tailored for synthetic biology

Much of the progress in developing our ability to successfully design genetic circuits with predictable dynamics has followed the strategy of molding biological systems to fit into conceptual frameworks used in other disciplines, most notably the engineering sciences. Because biological systems have fundamental differences from systems in these other disciplines, this approach is challenging and the insights obtained from such analyses are often not framed in a biologically-intuitive way. Here, we present a new theoretical framework for analyzing the dynamics of genetic circuits that is tailored towards the unique properties associated with biological systems and experiments. Our framework approximates a complex circuit as a set of simpler circuits, which the system can transition between by saturating its various internal components. These approximations are connected to the intrinsic structure of the system, so this representation allows the analysis of dynamics which emerge solely from the system's structure. Using our framework, we analyze the presence of structural bistability in a leaky autoactivation motif and the presence of structural oscillations in the Repressilator

A geometric and structural approach to the analysis and design of biological circuit dynamics: a theory tailored for synthetic biology

Much of the progress in developing our ability to successfully design genetic circuits with predictable dynamics has followed the strategy of molding biological systems to fit into conceptual frameworks used in other disciplines, most notably the engineering sciences. Because biological systems have fundamental differences from systems in these other disciplines, this approach is challenging and the insights obtained from such analyses are often not framed in a biologically-intuitive way. Here, we present a new theoretical framework for analyzing the dynamics of genetic circuits that is tailored towards the unique properties associated with biological systems and experiments. Our framework approximates a complex circuit as a set of simpler circuits, which the system can transition between by saturating its various internal components. These approximations are connected to the intrinsic structure of the system, so this representation allows the analysis of dynamics which emerge solely from the system's structure. Using our framework, we analyze the presence of structural bistability in a leaky autoactivation motif and the presence of structural oscillations in the Repressilator

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest

Fabrication of a Horizontal and a Vertical Large Surface Area Nanogap Electrochemical Sensor

Nanogap sensors have a wide range of applications as they can provide accurate direct detection of biomolecules through impedimetric or amperometric signals. Signal response from nanogap sensors is dependent on both the electrode spacing and surface area. However, creating large surface area nanogap sensors presents several challenges during fabrication. We show two different approaches to achieve both horizontal and vertical coplanar nanogap geometries. In the first method we use electron-beam lithography (EBL) to pattern an 11 mm long serpentine nanogap (215 nm) between two electrodes. For the second method we use inductively-coupled plasma (ICP) reactive ion etching (RIE) to create a channel in a silicon substrate, optically pattern a buried 1.0 mm × 1.5 mm electrode before anodically bonding a second identical electrode, patterned on glass, directly above. The devices have a wide range of applicability in different sensing techniques with the large area nanogaps presenting advantages over other devices of the same family. As a case study we explore the detection of peptide nucleic acid (PNA)−DNA binding events using dielectric spectroscopy with the horizontal coplanar device

Individual variation in body temperature and energy expenditure in response to mild cold

Department of Human Biology, Maastricht University, 6200 MD Maastricht, The Netherlands. [email protected] We studied interindividual variation in body temperature and energy expenditure, the relation between these two, and the effect of mild decrease in environmental temperature (16 vs. 22 degrees C) on both body temperature and energy expenditure. Nine males stayed three times for 60 h (2000-0800) in a respiration chamber, once at 22 degrees C and twice at 16 degrees C, in random order. Twenty-four-hour energy expenditure, thermic effect of food, sleeping metabolic rate, activity-induced energy expenditure, and rectal and skin temperatures were measured. A rank correlation test with data of 6 test days showed significant interindividual variation in both rectal and skin temperatures and energy expenditures adjusted for body composition. Short-term exposure of the subjects to 16 degrees C caused a significant decrease in body temperature (both skin and core), an increase in temperature gradients, and an increase in energy expenditure. The change in body temperature gradients was negatively related to changes in energy expenditure. This shows that interindividual differences exist with respect to the relative contribution of metabolic and insulative adaptations to cold

Addressable, "Packet-Based" Intercellular Communication through Plasmid Conjugation

We develop a system for implementing "packet-based" intercellular communication in an engineered bacterial population via conjugation. Our system uses gRNA-based identification markers that allow messages to be addressed to specific strains via Cas9-mediated cleavage of messages sent to the wrong recipient, which we show reduces plasmid transfer by four orders of magnitude. Integrase-mediated editing of the address on the message plasmid allows cells to dynamically update the message's recipients in vivo. As a proof-of-concept demonstration of our system, we propose a linear path scheme that would propagate a message sequentially through the strains of a population in a defined order

The upper critical field of filamentary Nb3Sn conductors

We have examined the upper critical field of a large and representative set of present multi-filamentary Nb3Sn wires and one bulk sample over a temperature range from 1.4 K up to the zero field critical temperature. Since all present wires use a solid-state diffusion reaction to form the A15 layers, inhomogeneities with respect to Sn content are inevitable, in contrast to some previously studied homogeneous samples. Our study emphasizes the effects that these inevitable inhomogeneities have on the field-temperature phase boundary. The property inhomogeneities are extracted from field-dependent resistive transitions which we find broaden with increasing inhomogeneity. The upper 90-99 % of the transitions clearly separates alloyed and binary wires but a pure, Cu-free binary bulk sample also exhibits a zero temperature critical field that is comparable to the ternary wires. The highest mu0Hc2 detected in the ternary wires are remarkably constant: The highest zero temperature upper critical fields and zero field critical temperatures fall within 29.5 +/- 0.3 T and 17.8 +/- 0.3 K respectively, independent of the wire layout. The complete field-temperature phase boundary can be described very well with the relatively simple Maki-DeGennes model using a two parameter fit, independent of composition, strain state, sample layout or applied critical state criterion.Comment: Accepted Journal of Applied Physics Few changes to shorten document, replaced eq. 7-