Journal: Nucleic Acids Research

Article Title: Incoherent merger network for robust ratiometric gene expression response

doi: 10.1093/nar/gkad087

Figure Lengend Snippet: The incoherent merger network and its genetic circuit implementation. ( A ) Merger network motif with inputs X and Y and disturbance R, which affects both P X and P Y with the same sign. Here, arrow ‘→’ denotes upregulation and ‘⊣’ represents downregulation. The loop formed by R, P X and P Y is an incoherent feedforward loop. ( B ) Genetic implementation of the network motif in (A). Here, X is the first input, which is a negative inducer that binds to repressor protein R X and prevents it from binding to DNA. Molecule m X is the mRNA of P X . Signaling molecule Y is the second input, which is a positive inducer that binds to activator protein A Y and allows DNA to be transcribed. Molecule m Y is the mRNA of P Y . The half disks in front of the gene coding region represent RBS sequences. In panel (A), R is any cellular resource that is equally required for the expression of P X and P Y , including transcriptional and translational resources. In the specific genetic implementation in (b), R is a translational resource, such as the ribosome. ( C ) Green colored plot is obtained from the reduced model in Supplementary Equation (S32) with parameters in where assumptions (A0) - (A2) are satisfied. Blue and red colored plots are obtained from the full model in Supplementary Equation (S10) with parameters in , in which perturbed R is 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\%$\end{document} of nominal R . 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\%$\end{document} change in R leads to only \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$5\%$\end{document} change in the output, so the system attenuates the change. ( D ) Relative \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\%$\end{document} error (= \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\vert P_{Y,{\rm }Nominal {\rm }R}-P_{Y, {\rm }Perturbed{\rm{ } R} \vert /P_{Y,{\rm }Nominal {\rm }R} \times 100 {\rm }(\%)$\end{document} ) of incoherent merger network and broken merging, in which perturbed R is 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\%$\end{document} of nominal R . In the broken merging, P X does not degrade P Y .

Article Snippet: Bacterial strain Escherichia coli Marionette DH10B (Addgene, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\#$\end{document} 108251) was used to construct and characterize genetic circuits.

Techniques: Binding Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Incoherent merger network for robust ratiometric gene expression response

doi: 10.1093/nar/gkad087

Figure Lengend Snippet: Performance and tunability of the incoherent merger network genetic implementation. ( A ) Genetic diagram of the incoherent merger network. This genetic circuit was created in three variants, depending on the choice of the RBS for sfGFP. Specifically, we chose TIRs as 1770, 4575, 8875 ( and Supplementary Figure S1 ). Both LacI and NahR regulators are endogenously expressed from the host E. coli Marionette strain . ( B ) Dose–response curves showing how GFP depends on Sal along with the reduced model in Supplementary Equation (S32) with parameter values in . ( C ) Dose–response curves showing how GFP depends on IPTG along with the reduced model in Supplementary Equation (S32) with parameter values in . ( D ) Response to the signal ratio Y / X . GFP/OD value of each circle is an average of three biologically independent replicates. ( E ) Response to the signal ratio Y / X for selected [Sal] and [IPTG] combinations in (D) that have the same ratio. ( F ) Distributions of output levels per cell for selected [Sal] and [IPTG] combinations that have the same ration (0.4). Coefficient of variation (CV) is 0.5374, 0.5025, 0.5007 for [IPTG] = 50, 75, 100 μM, respectively. All data were measured by flow cytometry when OD value was close to 0.054. Distributions of output levels per cell for all [IPTG] and [Sal] combinations are shown in Supplementary Figures S4 and S5. (B–F) were obtained with sfGFP’s TIR = 8875. ( G ) Tunability of the ratiometric sensor. Blue, green and red colored plots represent TIR = 8875, 4575, 1770, respectively. The temporal growth data and GFP expression with different TIRs can be found in Supplementary Figures S8– S10, respectively. Data in the line plots in panels (B) and (C) and data in the scatter plot in panel (D) and (G) represent mean values (±SD) of n = 3 biologically independent experiments.

Article Snippet: Bacterial strain Escherichia coli Marionette DH10B (Addgene, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\#$\end{document} 108251) was used to construct and characterize genetic circuits.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Determinants of metal import and specificity in a bacterial transporter

doi: 10.64898/2026.03.30.714904

Figure Lengend Snippet: (a) Schematic of our fluorescent reporter system that leverages both transcriptional and translational control to couple intracellular Mn 2+ concentration to fluorescence. (b) Example green fluorescence distributions (kernel density estimates) measured by flow cytometry for E. coli cells expressing WT DraNramp (yellow), M230A (orange), and two variants with low (N59D; teal) or no (D56A; dark gray) transport activity, alongside the pET28a empty vector (light gray). The x-axis is plotted on a logicle scale and events were gated to have similar cell sizes as measured by side scatter. (c) Dose-response curves normalized to the D56A data relating MnCl 2 concentration in the growth medium to fluorescence for the same variants as in (b), with overlaid fits to a sigmoid curve (resulting kinetic parameters listed in Supplementary Table 1). Error bars represent standard error of the mean from three replicates; sample raw distributions and an overview of the analysis are in . (d) At the bottom is a kernel density plot showing the distribution of cells in the first replicate of the evolution-guided library screen on the correlated green fluorescence (FITC-A) and side scatter area axes; approximate locations of the four sorted bins are overlaid. Above is the log-transformed enrichment of WT and two variants with intermediate (M230A) or no (D56G) Mn 2+ transport activity, highlighting how enrichment scores vary across bins for different variants. (e) Distribution of Mn 2+ activity scores for substitutions in the evolution-guided library across mutational depth. Scores range between ∼0 to ∼1 (representing no activity to WT-like levels of activity), with scores above 1 representing improved activity and scores below zero likely representing experimental noise. (f) Heatmap of Mn 2+ import scores for all variants with single mutation at positions with at least 5 measured variants, primarily from the binding-site library. White circles mark wildtype amino acids. Gray positions lack data.

Article Snippet: We transformed these ligation products into electrocompetent DH10B E. coli (GoldBio) and the requisite number of colonies were harvested from agar plates to bottleneck each subpool to ∼40X the subpool diversity to balance obtaining nearly every variant with attaining a reasonable barcode diversity.

Techniques: Control, Concentration Assay, Fluorescence, Flow Cytometry, Expressing, Activity Assay, Plasmid Preparation, Transformation Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Determinants of metal import and specificity in a bacterial transporter

doi: 10.64898/2026.03.30.714904

Figure Lengend Snippet: (a) MgKO, a Mg 2+ -auxotrophic strain of E. coli lacking any genetically encoded Mg 2+ transporters, can only survive in low Mg 2+ when rescued with a functional Mg 2+ transporter. (b) Example growth curves in LB supplemented with 1 mM MgSO 4 with 20 µM IPTG, showing robust growth for the Mg 2+ -transporting M230A variant (orange), no growth for the non-transporting WT DraNramp (gold), and intermediate growth for two replicates of the evolution-guided library (purple and teal). (c) Growth rates, normalized to WT in 1 mM supplemental MgSO 4 , measured across concentrations of Mg 2+ supplemented into LB medium. Rates are higher for M230A (right) compared to WT (left). Error bars represent standard error of the mean across three replicates. (d) Distribution of Mg 2+ import scores from the combined libraries, colored by whether the M230 position is mutated, with the region with scores above 2 (representing clear Mg 2+ import) in an inset. (e) Growth rates of isolated clones for a selected set of variants with no M230 mutation that have Mg 2+ import scores significantly higher than WT. M230A is included as a positive control. Stars represent significance thresholds from a series of Welch’s t-tests against the WT with a Benjamini-Hochberg correction (*: p<0.05). (f) Heatmaps of Mg 2+ import scores for all mutations to TM6 on the WT (top) and M230A (bottom) backgrounds. Black circles mark wildtype amino acids. Gray positions lack data. The 230 column is identical between heatmaps. Above the heatmaps is a snapshot of TM6 (PDB ID: 8E6N). The black asterisk approximates the bound Mn 2+ ; Mg 2+ may or may not bind the same site in the M230A variant. (g) Twenty-eight variants from the evolution-guided library with scores significantly outside the distribution defined by WT barcodes (FDR < 0.05 via Student’s t-test with a Benjamini-Hochberg correction). Variants were clustered based on which residues (color-coded by chemical property) were mutated using clustermap in seaborn. Arrows above and corresponding boxed columns highlight the positions of observed sequence couplings (purple: positions 54 and 275; green: positions 232 and 381/382). The asterisk represents a deletion of residues 125-127. (h) Scatterplot of Mg 2+ import scores for all single mutations present on both the WT (horizontal axis) and M230A (vertical axis) background. The gray diagonal line represents mutations with the same overall activity level on both backgrounds, while the horizontal gray lines represent the WT score (0.00 ± 0.04) or M230A score (7.29 ± 0.62). The black curve represents a fit to a site-independent sigmoid model, demonstrating how the mutations deviate considerably from an additive model even accounting for sigmoidal global epistasis. The shaded region around the sigmoidal curve represents a 99.7% confidence interval. A selection of mutations that improve Mg 2+ on their own are highlighted in teal, and several M230 mutations (which by definition have the same effect on each background) in orange.

Article Snippet: We transformed these ligation products into electrocompetent DH10B E. coli (GoldBio) and the requisite number of colonies were harvested from agar plates to bottleneck each subpool to ∼40X the subpool diversity to balance obtaining nearly every variant with attaining a reasonable barcode diversity.

Techniques: Functional Assay, Variant Assay, Isolation, Clone Assay, Mutagenesis, Positive Control, Sequencing, Activity Assay, Selection

Journal:

Article Title: Molecular and Biochemical Characterization of the Chromosome-Encoded Class A β-Lactamase BCL-1 from Bacillus clausii ▿

doi: 10.1128/AAC.00537-07

Figure Lengend Snippet: Localization of the blaBCL-1 gene in B. clausii. Total DNA from B. clausii ATCC 21537 (lanes 1) and NR (lanes 2) and from reference strain E. coli K-12 (lanes 3) was digested with I-CeuI and subjected to PFGE (A). DNA was transferred to a nylon membrane and hybridized successively with rrs (16 and 23S rRNA) (B) and blaBCL-1 (C) probes.

Article Snippet: TABLE 2. β-Lactam(s) a MIC (mg/ml) for b : B. clausii NR B. clausii ATCC 21537 E. coli DH10B(pAK) E. coli DH10B E. coli BL21(pCLA) E. coli BL21 Penicillin G 0.5 0.5 >512 32 128 2 Amoxicillin 0.5 1 >512 8 >512 1 Amoxicillin + CLA 0.12 0.12 32 4 2 1 Ticarcillin 8 8 >512 8 512 1 Ticarcillin + CLA 4 4 8 8 2 1 Piperacillin 16 16 8 4 32 1 Piperacillin + TZB 8 8 2 4 0.5 0.5 Cephalothin 8 8 64 4 16 1 Cefoxitin 32 16 16 8 2 0.5 Cefotaxime 16 16 0.12 0.06 0.06 0.06 Cefotaxime + CLA 16 16 0.06 0.06 0.06 0.06 Ceftazidime 128 128 0.5 0.12 0.12 0.06 Ceftazidime + CLA 128 128 0.5 0.12 0.06 0.06 Cefuroxime 128 128 16 8 2 0.5 Cefepime 256 128 0.06 0.06 0.06 0.06 Cefpirome 128 128 0.12 0.06 0.5 0.06 Cefpirome + CLA 8 8 0.06 0.06 0.06 0.06 Aztreonam >512 >512 0.12 0.12 0.06 0.06 Moxalactam 64 32 0.25 0.25 0.12 0.06 Imipenem 0.25 0.25 0.25 0.25 0.25 0.25 Open in a separate window a CLA, clavulanic acid at a fixed concentration of 2 μg/ml; TZB, tazobactam at a fixed concentration of 4 μg/ml. b B. clausii NR, E. coli DH10B(pAK), and E. coli BL2(pCLA) produced β-lactamase BCL-1.

Techniques: Membrane

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DH10B E. coli , NEB , Cat#C3019H.

Techniques: Recombinant, Protease Inhibitor, Mutagenesis, Clone Assay, Luciferase, Staining, Knock-Out, Marker, Software