e coli neb 10β  (New England Biolabs)

Journal: bioRxiv

Article Title: The evolution and mechanistic versatility of the bacterial NADH dehydrogenases type II

doi: 10.1101/2025.10.14.682086

Figure Lengend Snippet: Tree was obtained in RaxML (MSA 493 sequences, 402 sites). It is shown as midpoint rooted for visual purposes. TBE values are shown at selected nodes. Color of clades indicate the bacterial clades: gracilicutes (blue), terrabacteria (green) and DST (violet). Names of the most representative classes for each clade are given on the right. Orange circles indicate extant enzymes previously characterized: Sa: S. aureus , Ct: C. thermarum , Go: G. oxydans , Ec: E. coli , Mc: M. capsulatus , Mt: M. tuberculosis and Cg: C. glutamicum . The yellow circle depicts Anc3c sequence. Pink circles show the novel enzymes targeted here Bs: B. subtilis , To: T. oceani , Af: A. fermentans , Tf: T. fusca, Mr: M. ruestringensis , Mh: M. hydrothermalis and Xo: X. oceani . Fully annotated phylogeny in FigS1.

Article Snippet: E. coli NEB 10β and BL21 competent cells, ligase, and restriction enzymes were purchased from New England Biolabs (NEB, USA).

Techniques: 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

Journal: bioRxiv

Article Title: Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic crenarchaeon Sulfolobus islandicus

doi: 10.1101/2025.03.16.643552

Figure Lengend Snippet: Discovery of integration sites in S. islandicus M.16.4. (a) Computational workflow for the selection of candidate neutral integration sites. The pipeline was executed on the genome of S. islandicus M.16.4 for the endogenous Type I-A CRISPR-Cas system to identify 40-nucleotide protospacers with a ‘CCN’ PAM and at least 10 mismatches. 66 crRNAs targeting 21 intergenic regions were found suitable for gene integration. Available genomics, transcriptomics, and epigenomics data were integrated to guide the selection of 13 crRNAs targeting eight intergenic loci for characterization. (b) Multi-omics map for the integration sites located using the CRISPR-COPIES pipeline. Dashed vertical lines indicate the location of the identified integration sites along the chromosome of the M.16.4 strain. The green color highlights the sites chosen for characterization in this study. The height of solid yellow, green, and blue vertical lines represents the conservation score, log 10 (expression) of neighboring genes, and mean ClsN enrichment over approximately 150 nt mapped from REY15A data using BLAST, respectively. Red circles represent the origin of replications.

Article Snippet: The ligation mixture was transformed in NEB-10β E. coli competent cells and plated on LB agar plates supplemented with appropriate antibiotics.

Techniques: Selection, CRISPR, Expressing