e coli dh10b electrocompetent cells  (New England Biolabs)

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: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Efflux pump containing metagenomic DNA fragments from an aquarium confers tetracycline resistance. Microbroth dilution assay curves and calculated 50% inhibitory concentration (IC50) values for E. coli clones carrying the indicated metagenomic DNA fragments (TET1, TET3, or TET13). (A and B) tetracycline, (C and D) chloramphenicol, or (E and F) azithromycin. **** P < 0.0001, ** P < 0.005, * P < 0.05, n = 4 for all.

Article Snippet: The triplicate libraries were purified by PCR and a DNA cleanup kit, and the full 12 μL of purified library DNA was introduced into 25 μL of E. coli DH10B electrocompetent cells (New England Biolabs, C3020K) by electroporation at 1.8 kV in 0.1 mm cuvettes.

Techniques: Dilution Assay, Concentration Assay, Clone Assay

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Streptothricin resistance is conferred by the NTC1 metagenomic DNA fragment and satB . (A) The results of a microbroth dilution assay of E. coli clones grown in the presence of variable nourseothricin concentrations (NTC1, E. coli carrying the NTC1 metagenomic DNA fragment; stat , E. coli expressing the stat streptothricin acetyltransferase; satB , E. coli expressing the predicted satB open reading frame from the NTC1 fragment). (B) IC50 values calculated from dose-response curves. **** P < 0.0001, *** P < 0.0005, ** P < 0.005, n = 4 for all.

Article Snippet: The triplicate libraries were purified by PCR and a DNA cleanup kit, and the full 12 μL of purified library DNA was introduced into 25 μL of E. coli DH10B electrocompetent cells (New England Biolabs, C3020K) by electroporation at 1.8 kV in 0.1 mm cuvettes.

Techniques: Dilution Assay, Clone Assay, Expressing

Journal: bioRxiv

Article Title: Monitoring intracellular antibiotic concentrations in real-time using allosteric biosensors

doi: 10.64898/2026.02.05.704027

Figure Lengend Snippet: a-b , Schematic representation of the genetically encoded allosteric biosensors for (a) trimethoprim and (b) tetracyclines. c , Overlay of DHFR in the trimethoprim (Trim)-bound (blue-gray) state and unbound state (beige), in cartoon presentation (left) with a close-up of the Trim binding site (right). The “Met20 loop” in the apo- and holo-form is highlighted in bright yellow and dark blue, respectively; the active site Asp27 is highlighted in shades of green; the insertion site of cpEGFP in shades of violet. In the close-up view, hydrophobic residues Leu20 (corresponding to the namesake Met20 in E. coli ) and Trp22 in the “Met20 loop” that flip into the binding pocket upon Trim-binding are displayed as spheres in the holo-structure. d , Excitation spectra of trimethoprim sensor TrimFABS with and without Trim in the presence of 250 μM NADPH. e , Emission spectra of TrimFABS with and without Trim and excitation at 405 nm (blue) or 467 nm (orange) in the presence of 250 μM NADPH. f , Trim dose-response curves of TrimFABS in the presence of 250 μM NADPH, showing emission at 550 nm with excitation at 405 nm or 497 nm (left y-axis) and the combined ratiometric readout (gray points, ratio of 550 nm emission signal with 497 nm and 405 nm; right y -axis). A Trim dose-response curve of TrimFABS without NADPH, excited at 497 nm, is shown for comparison (red). Data points are pooled from n=6 independent dilution series with six repeated measurements for each concentration at steady state. g , NADPH dose-response curves of TrimFABS in the presence of 1.25 mM Trim, showing emission at 550 nm with excitation at 405 nm and 497 nm (left y-axis) and the combined ratiometric readout (ratio of 550 nm emission signal with 497 nm and 405 nm; right y -axis). A NADPH dose-response curve of TrimFABS without Trim at 497 nm excitation is shown for comparison (red). Data points represent pooled from n=6 independent dilution series with six repeated measurements for each concentration at steady state. h , Overlay of TetR in the anydrotetracycline (aTc)-bound (turquoise) and unbound state (beige), in cartoon presentation (left) and a close-up of the binding site (right). The region (residues 145-149) where cpEGFP* is inserted is highlighted in light and dark violet in the aTc-unbound and -bound forms, respectively. The crucial Glu147 (shown as stick) and magnesium ion (green sphere) stabilizing tetracycline binding via water molecules (small red spheres) are indicated. i , Excitation spectra of tetracycline sensor TetFABS with and without aTc. j , Emission spectra of TetFABS with and without aTc and excitation at 405 nm (blue lines) or 467 nm (orange lines). k , aTc dose-response curves of TrimFABS emission at 550 nm with excitation at 405 nm or 497 nm (left y-axis) and the combined ratiometric readout combined ratiometric readout (gray points, ratio of 550 nm emission signal with 497 nm and 405 nm; right y -axis). Data points represent pooled data from n=3 independent dilution series with six repeated measurements for each concentration at steady state. l , Affinity (K d ) of different antibiotics of the class of tetracyclines to TrimFABS. Shown are mean (circles) and 95% confidence intervals (lines) obtained from fits of dose-response curves, from n=3-6 independent replicas for each condition (see ).

Article Snippet: Resulting reaction products were purified, eluted in 30 μL of milliQ water, transformed into 50 μL of E. coli MegaX DH10B T1 electrocompetent cells (Invitrogen), and transferred into liquid LB medium supplemented with ampicillin (250 μg mL -1 ).

Techniques: Binding Assay, Comparison, Concentration Assay