Generative frame interpolation enhances tracking of biological objects in time-lapse microscopy.

Object tracking in microscopy videos is crucial for understanding biological processes. While existing methods often require fine-tuning tracking algorithms to fit the image dataset, here we explored an alternative paradigm: augmenting the image time-lapse dataset to fit the tracking algorithm. To test this approach, we evaluated whether generative video frame interpolation can augment the temporal resolution of time-lapse microscopy and facilitate object tracking in multiple biological contexts.

A library of reporters of the global regulators of gene expression in .

The topology of the transcription factor network (TFN) of is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose.

Hardware, Software, and Wetware Codesign Environment for Synthetic Biology.

Synthetic biology is the process of forward engineering living systems. These systems can be used to produce biobased materials, agriculture, medicine, and energy. One approach to designing these systems is to employ techniques from the design of embedded electronics.

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration.

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli.

Buildout and integration of an automated high-throughput CLIA laboratory for SARS-CoV-2 testing on a large urban campus.

In 2019, the first cases of SARS-CoV-2 were detected in Wuhan, China, and by early 2020 the first cases were identified in the United States. SARS-CoV-2 infections increased in the US causing many states to implement stay-at-home orders and additional safety precautions to mitigate potential outbreaks. As policies changed throughout the pandemic and restrictions lifted, there was an increase in demand for COVID-19 testing which was costly, difficult to obtain, or had long turn-around times.

Genetic circuit design automation with Cello 2.0.

Cells interact with their environment, communicate among themselves, track time and make decisions through functions controlled by natural regulatory genetic circuits consisting of interacting biological components. Synthetic programmable circuits used in therapeutics and other applications can be automatically designed by computer-aided tools. The Cello software designs the DNA sequences for programmable circuits based on a high-level software description and a library of characterized DNA parts representing Boolean logic gates.

Transcription closed and open complex formation coordinate expression of genes with a shared promoter region.

Many genes are spaced closely, allowing coordination without explicit control through shared regulatory elements and molecular interactions. We study the dynamics of a stochastic model of a gene-pair in a head-to-head configuration, sharing promoter elements, which accounts for the rate-limiting steps in transcription initiation. We find that only in specific regions of the parameter space of the rate-limiting steps is orderly coexpression exhibited, suggesting that successful cooperation between closely spaced genes requires the coevolution of compatible rate-limiting step configuration.

Estimating RNA numbers in single cells by RNA fluorescent tagging and flow cytometry.

Estimating the statistics of single-cell RNA numbers has become a key source of information on gene expression dynamics. One of the most informative methods of in vivo single-RNA detection is MS2d-GFP tagging. So far, it requires microscopy and laborious semi-manual image analysis, which hampers the amount of collectable data.

Chromosome and plasmid-borne P promoters differ in sensitivity to critically low temperatures.

Temperature shifts trigger genome-wide changes in Escherichia coli's gene expression. We studied if chromosome integration impacts on a gene's sensitivity to these shifts, by comparing the single-RNA production kinetics of a P promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar.

SCIP: a single-cell image processor toolbox.

Summary: Each cell is a phenotypically unique individual that is influenced by internal and external processes, operating in parallel. To characterize the dynamics of cellular processes one needs to observe many individual cells from multiple points of view and over time, so as to identify commonalities and variability. With this aim, we engineered a software, 'SCIP', to analyze multi-modal, multi-process, time-lapse microscopy morphological and functional images.

The precision of the symmetry in Z-ring placement in Escherichia coli is hampered at critical temperatures.

Cell division in Escherichia coli is morphologically symmetric due to, among other things, the ability of these cells to place the Z-ring at midcell. Studies have reported that, at sub-optimal temperatures, this symmetry decreases at the single-cell level, but the causes remain unclear. Using fluorescence microscopy, we observe FtsZ-GFP and DAPI-stained nucleoids to assess the robustness of the symmetry of Z-ring formation and positioning in individual cells under sub-optimal and critical temperatures.

Temperature-dependence of the single-cell variability in the kinetics of transcription activation in Escherichia coli.

From in vivo single-cell, single-RNA measurements of the activation times and subsequent steady-state active transcription kinetics of a single-copy Lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of the inducer (IPTG) from the media, following temperature shifts. For this, for temperature shifts of various degrees, we obtain the distributions of transcription activation times as well as the distributions of intervals between consecutive RNA productions following activation in individual cells. We then propose a novel methodology that makes use of deconvolution techniques to extract the mean and the variability of the distribution of intake times.

Temperature-Dependent Model of Multi-step Transcription Initiation in Escherichia coli Based on Live Single-Cell Measurements.

Transcription kinetics is limited by its initiation steps, which differ between promoters and with intra- and extracellular conditions. Regulation of these steps allows tuning both the rate and stochasticity of RNA production. We used time-lapse, single-RNA microscopy measurements in live Escherichia coli to study how the rate-limiting steps in initiation of the Plac/ara-1 promoter change with temperature and induction scheme.

Dissecting the stochastic transcription initiation process in live Escherichia coli.

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average.

Increased cytoplasm viscosity hampers aggregate polar segregation in Escherichia coli.

In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub-optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location.

Kinetics of the cellular intake of a gene expression inducer at high concentrations.

From in vivo single-event measurements of the transient and steady-state transcription activity of a single-copy lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of its inducer (IPTG) from the media. We show that the empirical data are well-fit by a model of intake assuming a bilayer membrane, with the passage through the second layer being rate-limiting, coupled to a stochastic, sub-Poissonian, multi-step transcription process. Using this model, we show that for a wide range of extracellular inducer levels (up to 1.

Single-cell kinetics of a repressilator when implemented in a single-copy plasmid.

Synthetic genetic clocks, such as the Elowitz-Leibler repressilator, will be key regulatory components of future synthetic circuits. We constructed a single-copy repressilator (SCR) by implementing the original repressilator circuit on a single-copy F-plasmid. After verifying its functionality, we studied its behaviour as a function of temperature and compared it with that of the original low-copy-number repressilator (LCR).

Robustness of the division symmetry in Escherichia coli and functional consequences of symmetry breaking.

The morphological symmetry of the division process of Escherichia coli is well-known. Recent studies verified that, in optimal growth conditions, most divisions are symmetric, although there are exceptions. We investigate whether such morphological asymmetries in division introduce functional asymmetries between sister cells, and assess the robustness of the symmetry in division to mild chemical stresses and sub-optimal temperatures.

In vivo kinetics of segregation and polar retention of MS2-GFP-RNA complexes in Escherichia coli.

The cytoplasm of Escherichia coli is a crowded, heterogeneous environment. From single cell live imaging, we investigated the spatial kinetics and heterogeneities of synthetic RNA-protein complexes. First, although their known tendency to accumulate at the cell poles does not appear to introduce asymmetries between older and newer cell poles within a cell lifetime, these emerge with cell divisions.

Effects of temperature on the dynamics of the LacI-TetR-CI repressilator.

We studied the behaviour of the repressilator at 28 °C, 30 °C, 33 °C, and 37 °C. From the fluorescence in each cell over time, we determined the period of oscillations, the functionality (fraction of cells exhibiting oscillations) and the robustness (fraction of expected oscillations that occur) of this circuit. We show that the oscillatory dynamics differs with temperature.

In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter.

Using a single-RNA detection technique in live Escherichia coli cells, we measure, for each cell, the waiting time for the production of the first RNA under the control of PBAD promoter after induction by arabinose, and subsequent intervals between transcription events. We find that the kinetics of the arabinose intake system affect mean and diversity in RNA numbers, long after induction. We observed the same effect on Plac/ara-1 promoter, which is inducible by arabinose or by IPTG.