Synchronous long-term oscillations in a synthetic gene circuit

Synthetically engineered genetic circuits can perform a wide variety of tasks but are generally less accurate than natural systems. Here we revisit the first synthetic genetic oscillator, the repressilator1, and modify it using principles from stochastic chemistry in single cells. Specifically, we sought to reduce error propagation and information losses, not by adding control loops, but by simply removing existing features. We show that this modification created highly regular and robust oscillations. Furthermore, some streamlined circuits kept 14 generation periods over a range of growth conditions and kept phase for hundreds of generations in single cells, allowing cells in flasks and colonies to oscillate synchronously without any coupling between them. Our results suggest that even the simplest synthetic genetic networks can achieve a precision that rivals natural systems, and emphasize the importance of noise analyses for circuit design in synthetic biology.Some work was performed at the Harvard Medical School Microfluidics Facility and the Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network supported by NSF award ECS-0335765. LPT acknowledges fellowship support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Fonds de recherche du Québec – Nature et technologies. This work was supported by NIH Grant GM095784 and NSF Award 1137676.This is the author accepted manuscript. The final version is available from Nature via https://www.nature.com/nature/journal/v538/n7626/full/nature19841.htm

Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells

AbstractKnowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique—spatial intensity distribution analysis (SpIDA)—that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP

Synchronous long-term oscillations in a synthetic gene circuit.

Synthetically engineered genetic circuits can perform a wide variety of tasks but are generally less accurate than natural systems. Here we revisit the first synthetic genetic oscillator, the repressilator, and modify it using principles from stochastic chemistry in single cells. Specifically, we sought to reduce error propagation and information losses, not by adding control loops, but by simply removing existing features. We show that this modification created highly regular and robust oscillations. Furthermore, some streamlined circuits kept 14 generation periods over a range of growth conditions and kept phase for hundreds of generations in single cells, allowing cells in flasks and colonies to oscillate synchronously without any coupling between them. Our results suggest that even the simplest synthetic genetic networks can achieve a precision that rivals natural systems, and emphasize the importance of noise analyses for circuit design in synthetic biology.Some work was performed at the Harvard Medical School Microfluidics Facility and the Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network supported by NSF award ECS-0335765. LPT acknowledges fellowship support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Fonds de recherche du Québec – Nature et technologies. This work was supported by NIH Grant GM095784 and NSF Award 1137676.This is the author accepted manuscript. The final version is available from Nature via https://www.nature.com/nature/journal/v538/n7626/full/nature19841.htm

Microfluidics and single-cell microscopy to study stochastic processes in bacteria

Bacteria have molecules present in low and fluctuating numbers that randomize cell behaviors. Understanding these stochastic processes and their impact on cells has, until recently, been limited by the lack of single-cell measurement methods. Here, we review recent developments in microfluidics that enable following individual cells over long periods of time under precisely controlled conditions, and counting individual fluorescent molecules in many cells. We showcase discoveries that were made possible using these devices in various aspects of microbiology, such as antibiotic tolerance/persistence, cell-size control, cell-fate determination, DNA damage response, and synthetic biology

The integrin-ligand interaction regulates adhesion and migration through a molecular clutch.

Adhesive and migratory behavior can be cell type, integrin, and substrate dependent. We have compared integrin and substrate differences using three integrin receptors: α5β1, α6β1, and αLβ2 expressed in a common cell type, CHO.B2 cells, which lack integrin α subunits, as well as in different cell types that express one or more of these integrins. We find that CHO.B2 cells expressing either α6β1 or αLβ2 integrins migrate and protrude faster and are more directionally persistent on laminin or ICAM-1, respectively, than CHO.B2 cells expressing α5β1 on fibronectin. Despite rapid adhesion maturation and the presence of large adhesions in both the α6β1- and αLβ2-expressing cells, they display robust tyrosine phosphorylation. In addition, whereas myosin II regulates adhesion maturation and turnover, protrusion rates, and polarity in cells migrating on fibronectin, surprisingly, it does not have comparable effects in cells expressing α6β1 or αLβ2. This apparent difference in the integration of myosin II activity, adhesion, and migration arises from alterations in the ligand-integrin-actin linkage (molecular clutch). The elongated adhesions in the protrusions of the α6β1-expressing cells on laminin or the αLβ2-expressing cells on ICAM-1 display a novel, rapid retrograde flux of integrin; this was largely absent in the large adhesions in protrusions of α5β1-expressing cells on fibronectin. Furthermore, the force these adhesions exert on the substrate in protrusive regions is reduced compared to similar regions in α5-expressing cells, and the adhesion strength is reduced. This suggests that intracellular forces are not efficiently transferred from actomyosin to the substratum due to altered adhesion strength, that is, avidity, affinity, or the ligand-integrin-actin interaction. Finally, we show that the migration of fast migrating leukocytes on fibronectin or ICAM-1 is also largely independent of myosin II; however, their adhesions are small and do not show retrograde fluxing suggesting other intrinsic factors determine their migration differences

Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator

Abstract: Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings

HL-60 migration on FN or ICAM-1.

<p>(A) Migration tracks of HL-60 cells on the indicated substrate over 1 hour were translated to a common origin and marked with a different color. Scale Bar  = 100 µm. (B) The speed and directionality were calculated and plotted (n = 36, 39 cell tracks, respectively). At least three independent experiments were quantified. P value  = 3×10⁻⁷.</p