e coli mc1061  (ATCC)

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Nanobody-functionalised silica particles bind selectively to target bacteria. i) Graphical illustration of particle–bacteria binding strategy. The cysteine group on the nanobody is bound to a PEG11 linker via a maleimide bond which, in turn, is bound to streptavidin-functionalised 2.7 μm silica particles via a biotin bond. ii) Particle–bacteria binding after mixing for 30 min in a 1.5 ml Eppendorf and imaging in a 96-well plate in the phase contrast channel or iii) fluorescence channel. White arrow highlights a silica particle, blue arrow highlights a bacterium. a) Sybody-F1 (SbF1) functionalised particles bound to S. aureus GFP. b) Nanobody 01 (Nb01) functionalised particles bound to E. coli MC1061 expressing mScarlet and naturally expressing OmpA-short. c) Nanobody 41 (Nb41) functionalised particles bound to E. coli MC1061 Δ ompA expressing mNeonGreen and OmpA-long. Scale bar = 5 μm.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Bacteria, Binding Assay, Imaging, Fluorescence, Expressing

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Single species growth upon bacterial binding to sCAPA deposited particles. a) Graphical illustration of deposition and binding assay. Particles are first deposited with sCAPA, and the template is then transferred to a Petri dish and filled with BSA to cover the PDMS. Bacteria are then added to bind to the particles. Unbound bacteria are washed out with fresh PBS using a pipette. PBS is then replaced with agar media, before placing the petri dish under a microscope to image cell growth. b) Representative fluorescence images of S. aureus JE2 GFP bound to SbF1 deposited particles. i) Cells bound at t = 40 min after adding tryptone soy agar. ii) Cells growing after t = 140 min. iii) Cells growing at t = 260 min where colonies begin to merge. Scale bar = 50 μm. c) E. coli MC1061 mScarlet bound to Nb01 deposited particles. i) Cy3 image of cell binding at t = 40 min. ii) Phase contrast image of cell growth after t = 260 min. Cy3 fluorescence not visible at exponential phase due to weak fluorescence. iii) Cell growth at t = 310 min where colonies begin to merge. Scale bar = 50 μm. d) Binarised images in c used during image quantification. Binarised colony in white, red circles represent a circle with an equivalent area as overlaying colony to which a ‘colony radius’ is attributed. i) Cells bound at t = 40 min after adding tryptone soy agar, ii) colony formation after 260 min growth, iii) colonies begin to merge at 310 min. e) Quantification of the fraction of traps with observed microbial growth. Bars and errors represent mean and standard error of the mean, data points represent different fractions measured for each field of view for 3 separate templates for each bacterial strain. f) Distribution of number of bacteria bound in each trap as determined by particle localisation image analysis on ×60 magnification images of templates in PBS. Despite the particles all being the same size, the average number of bound bacteria to each particle notably varies. Mean number of bound bacteria and standard error of the mean displayed in top right. g) Distribution in time taken for individual colonies to reach a radius of 15 μm. Data points represent single growing colonies. Distributions represent all data for biological triplicates. Black lines represent mean and variance. h) Growth of indicated bacterial strains in liquid tryptone soy broth measured by optical density at 600 nm. Error bars represent standard error of the mean of 3 biological repeats and the black dotted line represents fit to a Gompertz growth law.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Bacteria, Transferring, Microscopy, Fluorescence

Journal: Lab on a Chip

Article Title: Controlling spatial structure in minimal microbial communities by sequential capillary assembly

doi: 10.1039/d6lc00040a

Figure Lengend Snippet: Cell binding of microbial pairs with designed spatial structure. a–c) Representative images of selectively bound and growing cells across different time points. a) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in a sodium chloride lattice structure corresponding to Moran's I = −1. b) E. coli mScarlet (OmpA-short) and E. coli mNeonGreen (OmpA-long) in 8 × 8 patchy lattice structure corresponding to Moran's I = 0.75. c) S. aureus GFP and E. coli mScarlet (OmpA-long) in 1 : 24 ratio square lattice. i) Merged Cy3 and FITC image in PBS media, ii) cell growth after exchanging PBS with tryptone soy agar and growing at 37 °C. Distance between traps in all lattices is 25 μm. Time stamp in hours:mins after exchanging PBS for agar. Scale bar = 50 μm. d) Comparison of ideal vs. measured cell binding for spatial structures in a–c. Bars represent mean of 4 independent templates. e) Fraction of growing cells for sum of green and red cells. Data points represent measured growing fraction in individual microscope fields of view, for 4 independent templates. Error bars represent standard error of the mean.

Article Snippet: For E. coli , we developed chromosomally fluorescent strains expressing mNeonGreen or mScarlet proteins in E. coli MC1061 (ATCC 37493, obtained from the lab of Markus Seeger).

Techniques: Binding Assay, Comparison, Microscopy

Journal: bioRxiv

Article Title: Simple high-throughput encoding of deep mutational scanning libraries by oligo-based Golden Gate assembly

doi: 10.1101/2025.07.16.665225

Figure Lengend Snippet: (A): Residues encompassing the CDR regions were targeted for mutation (red). The nanobody CDS was split into six parts to facilitate effective synthesis of the CDRs (parts 2, 3, and 5) as separate oligo pools. Part boundaries were determined by suitability of overhangs (purple/pink) for Golden Gate assembly via BsaI restriction-enzyme digest. The nanobody CDS termini (parts 1 and 6) were synthesised with an internal counter-selection cassette ( ccdB ) flanked by BsaI recognition sites. The fragment was cloned into a phagemid (F1 ori) destination vector, with 5’ pelB and 3’ M13 phage pIII fusions (yellow) to facilitate phage expression upon assembly with the remaining CDS parts (2-5). These parts were synthesised with flanking BsaI sites and cloned into pUC19-derived vectors prior to Golden Gate assembly. Variant part generation (B): Oligos of the three parts representing the CDRs (2, 3, 5) were designed as single or dual NNK-codon containing libraries and synthesised as separate forward (FWD) and reverse complement (RC) oligo pools (oPools). Each oligo contained a unique 5’ Golden Gate overhang, which after 5’ phosphorylation and duplexing via heat-cool cycles, enabled their immediate ligation into overhang-matched pre-digested vectors. The ligation products were immediately purified and electro-transformed to yield separate single and dual variant libraries for each part. Golden Gate assemblies (C): By substituting wild-type parts with variant library parts during Golden Gate assembly, seven nanobody CDS variant libraries were generated. Six of these formed the dual DMS library; ≤2 substitutions total, either inter-or intra-CDR. The seventh library was assembled from the dual intra-CDR parts to yield a high diversity combinatorial library; ≤ 6 substitutions total, ≤2 intra-CDR. The assembled libraries were electro-transformed into NEB 5-alpha F’Iq E. coli . Variant screening (D): All bacterial libraries were cultured and infected with M13KO7 helper phage to produce nanobody-pIII fused phage. This recombinant phage was then screened against an immobilised antigen (PEP-1) before recovery via infection and plating of fresh E. coli . This panning process was repeated for multiple rounds.

Article Snippet: Purified ligation products were transformed into fresh, electrocompetent E. coli MC1061 cells via MicroPulser Electroporator (Bio-Rad, USA) according to the manufacturer’s manual, before being entirely plated across several large (140 mm) selective LB agar plates, with ∼100 μL reserved for colony quantification via plated serial dilution.

Techniques: Mutagenesis, Selection, Clone Assay, Plasmid Preparation, Expressing, Derivative Assay, Variant Assay, Phospho-proteomics, Ligation, Purification, Transformation Assay, Generated, Cell Culture, Infection, Recombinant

Journal: bioRxiv

Article Title: Simple high-throughput encoding of deep mutational scanning libraries by oligo-based Golden Gate assembly

doi: 10.1101/2025.07.16.665225

Figure Lengend Snippet: ( A ) Nanobody variants were selected with the highest-ranking enrichment scores from both the dual site (3 variants) and combinatorial libraries (1 variant). Nanobodies were expressed in E. coli , purified with MabSelect™ PrismA resin, and run by SDS-PAGE using a 4-20% gradient gel. The wild-type variant was expressed in parallel. ( B ) Nanobody affinity was measured by ELISA against PEP-1 peptide coated plates. Nanobodies were diluted from a starting concentration of 15 μg/mL, with the anti-EGFR nanobody 7D12 used as a negative control. EC50 values were obtained by fitting of 5-parameter logistic (10.1, 10.6, 10.m1) or 4-parameter logistic models (WT).

Article Snippet: Purified ligation products were transformed into fresh, electrocompetent E. coli MC1061 cells via MicroPulser Electroporator (Bio-Rad, USA) according to the manufacturer’s manual, before being entirely plated across several large (140 mm) selective LB agar plates, with ∼100 μL reserved for colony quantification via plated serial dilution.

Techniques: Variant Assay, Purification, SDS Page, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control

Journal: The EMBO Journal

Article Title: Proton conductance by human uncoupling protein 1 is inhibited by purine and pyrimidine nucleotides

doi: 10.1038/s44318-025-00395-3

Figure Lengend Snippet: Reagents and tools table

Article Snippet: E. coli strain MC1061 , ATCC , ATCC number: 47035.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Software