Journal: bioRxiv

Article Title: Period Doubling Bifurcations in a Forced Cell-Free Genetic Oscillator

doi: 10.1101/2021.12.15.472802

Figure Lengend Snippet: Genetic oscillator circuit and experimental setup. (a) Simple and (b) detailed circuit diagram of the synthetic genetic oscillator consisting of a negative feedback loop where σ 28 acts as activator and TetR acts as repressor. Our four-variable ODE model ( – ) considers the concentrations of the two proteins and their respective mRNAs. Protein expression is monitored via co-expression of fluorescent reporters mVenus and mTurquoise2, respectively. We can perturb the system either by inactivating existing TetR by induction with aTc, or by adding purified TetR. (c) A microfluidic ring reactor was used to maintain the reaction out-of-equilibrium by periodically exchanging a fraction of the reactor volume with fresh reagents. By switching between different input reagents, the reactions can be exposed to an arbitrary series of inputs. (d) Transfer functions of the two promoters determined by titrating the regulator protein in bulk. Fitted Hill parameters with 68% confidence intervals are K h = 2.2 ± 0.2 nM, n h = 2.1 ± 0.3, K a = 115 ± 6 nM n a = 3.4 ± 0.6. N.F.I.: Normalized Fluorescence Intensity.

Article Snippet: The experimental setup used to control the microfluidic device and image acquisition was custom built around an Olympus IX81 inverted epifluorescence microscope equipped with a motorized stage, fluorescence light engine (Lumencor SOLA SE II 365), camera (Andor iXon3 DU888), and filters (CFP: 438-25/458/483-32, YFP: 500-20/515/535-30, RFP: 559-34/588/609-34, GFP: 472-30/495/520-34).

Techniques: Expressing, Purification, Fluorescence

Journal: bioRxiv

Article Title: Single-cell transcriptome imaging reveals conserved and virulence-linked phenotypic states in bacteria

doi: 10.64898/2026.02.01.703127

Figure Lengend Snippet: ( A-B ) Coefficient of variation (CV) and Tails Expression Ratio (TER) values plotted against mean expression for each gene–condition datapoint with expression in a sufficient number of cells (n = 13,006). Individual datapoints are shown in gray, and those passing the heterogeneity threshold based on integrated CV and TER metrics (EVI score) are highlighted in pink. Mean expression values are reported in arbitrary fluorescence units. The fitted trend and 95% confidence intervals are shown as a black line and gray shading, respectively. ( C ) EVI distributions per condition (EVI calculated with datapoints from all conditions). The threshold EVI demarking the top 1% of variable data points is shown as a dashed line (EVI = 2.7). The median EVI of each condition is shown as a horizontal black line. All gene-condition data points are shown as gray points with several genes of interest highlighted as colored triangles. ( D ) Raw par 2 FISH data shown over randomly selected cells for several hypervariable genes. ( E ) Independent validation using promoter reporters for three genes ( ompC , aceB , and rcsA , the activator of gmd ); additional reporters shown in fig. S3. Scale bar, 5 µm. Fluorescence intensity (D-E) is displayed with the inferno color map, as shown in the reference bar within the figure.

Article Snippet: Fluorophore excitation was provided by a SOLA III FISH Light Engine (Lumencor) in combination with standard fluorescence excitation/emission filter cubes for DAPI, FITC (Alexa Fluor 488), TRITC (Atto 550), and Cy5 (Atto 647).

Techniques: Expressing, Fluorescence, Biomarker Discovery