e coli neb 10β  (New England Biolabs)

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Overview of High Complexity Golden Gate Assembly (HC-GGA) design and assembly. A) The coliphage T7 genome was divided into 21 fragments for modular assembly. Genes containing native amber (TAG) stop codons are shown in orange, and the major capsid protein gene (gp10A) is highlighted in blue. B) These fragments were generated by PCR from either BsmBI-domesticated T7 genomic DNA or synthetic gBlocks, assembled using HC GGA into a complete genome, and transformed into E. coli NEB 10β cells by electroporation. Individual plaques were isolated and sequenced to confirm successful genome reconstruction. C) For the Amber Free (AF) and Amber Free/NanoLuc (AF/NL) variants, selected fragments (F2, F6, F9, F10, F14, F19, and F21) were replaced with synthetic versions. In the AF variant, all native amber codons were recoded to ocher (TAA) codons to prevent unintended incorporation of noncanonical amino acids. In the AF/NL variant, an amber codon was inserted at the end of gp10A to enable site-specific incorporation of L-homopropargylglycine, and a NanoLuc luciferase gene was inserted downstream of gp10B under a T7 promoter. Upon infection of E. coli by the engineered phage, the host expresses the NanoLuc protein. After lysis, NanoLuc interacts with its substrate to produce luminescence. This signal is only generated if the phage successfully binds to, concentrates, and infects its E. coli host, thereby enabling sensitive and specific detection of viable bacteria in water samples. Figure created in Biorender.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Generated, Transformation Assay, Electroporation, Isolation, Variant Assay, Luciferase, Infection, Lysis, Bacteria

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic for phage conjugation to nanoparticles via click chemistry. Cobalt-containing azide functionalized magnetic nanoparticles were conjugated to phages containing an alkyne-modified noncanonical amino acid (L-HPG) incorporated into the major capsid protein using a copper-mediated cycloaddition resulting in magnetized phages which could be used to capture and detect their host E. coli . Figure created in Biorender.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Conjugation Assay, Modification

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic overview of the bacteriophage-based assay for detection of E. coli in drinking water. A) An overnight culture of E. coli strain ECOR 13 is grown and then inoculated into fresh media. B) The culture is monitored by measuring optical density at 600 nm (OD 600 ) to ensure an appropriate growth phase, followed by cooling on ice. C) Tap water is filtered through a 0.22 μm membrane and stored at 4 °C to simulate drinking water conditions. D) The E. coli culture is serially diluted using the filtered tap water to prepare test samples with defined bacterial concentrations. E) Phage-coated magnetic nanoparticles are added to each inoculated sample and incubated for 30 min to allow binding and infection. F) Magnetized phages and any bound E. coli are separated using a magnetic rack. G) A luminescent substrate specific to NanoLuc is added to the isolated complexes. H) Upon lysis of infected E. coli , NanoLuc is released and reacts with the substrate to produce a luminescent signal. I) Luminescence is measured using a plate reader, providing a quantitative readout of viable E. coli presence in the sample. Figure created in Biorender.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Membrane, Incubation, Binding Assay, Infection, Isolation, Lysis

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Magnetized phages were used to detect E. coli (ECOR 13) in 100 mL drinking water samples. Data points represent the average of triplicates and error bars represent standard deviations. The average signal of the negative controls, consisting of uninoculated samples (0 CFU) are represented by a dashed line with the standard deviations represented by dotted lines.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques:

Journal: ACS Synthetic Biology

Article Title: Recoded Bacteriophage Genome for Bio-Orthogonal-Enabled Concentration and Detection of E. coli in Drinking Water

doi: 10.1021/acssynbio.5c00665

Figure Lengend Snippet: Schematic overview of the genetic engineering workflow for constructing modified T7 bacteriophage genomes. a) PCR amplification of 21 fragments from the BsmBI-domesticated T7 genome. b) SPRI-based size selection and nucleic acid purification, followed by validation of fragment size and homogeneity via gel electrophoresis and quantification using Qubit. c) Assembly of fragments using Golden Gate Assembly with BsmBI, cycled at 42 °C for 5 min and 16 °C for 5 min over 15 cycles. d) Electroporation of 1 μL of the circularized genome into competent E. coli 10-beta cells, followed by 1.5 h of recovery at 37 °C in stable outgrowth media. e) Dilution plating with E. coli host, isolation of plaques, and whole-genome sequencing to confirm successful assembly and modification.

Article Snippet: A portion of the assembly reactions (1 μL) were transformed into E. coli NEB 10β (C3020, NEB) cells by electroporation utilizing a BioRad GenePulser Xcell Microbial System under standard conditions (1.8 kV, 25 μF, 200 Ω).

Techniques: Modification, Amplification, Size Selection, Nucleic Acid Purification, Biomarker Discovery, Nucleic Acid Electrophoresis, Electroporation, Isolation, Sequencing

Journal: bioRxiv

Article Title: The evolution of allostery in a protein family

doi: 10.1101/2025.06.20.660748

Figure Lengend Snippet: a. Free energy heat maps for all PDZ domain interactions. Dashes indicate wild-type residues. Residue numbering follows UniProt protein sequence annotation. b. Correlations with independent energy measurements from Salinas and Ranganathan 2018 . BindingPCA free energies are from a single model; error bars indicate 95% confidence intervals from a Monte Carlo simulation approach (n = 10 experiments). Pearson’s R is shown together with a linear model fit with 95% confidence intervals shown with green shading. c. PDZ domain structures colored by median free energy changes (ΔΔG) at each residue position. For each domain, the first structure highlights median ΔΔG f values in the core residues. The second and third structures are oriented towards the ligand-binding interface, displaying surface coloring based on ΔΔG f and ΔΔG b , respectively.

Article Snippet: At each step, gibson products were dialyzed, concentrated to 5μL using a SpeedVac machine, and transformed into NEB 10β competent E. coli cells according to the manufacturer’s protocol.

Techniques: Residue, Sequencing, Ligand Binding Assay