t7 express high efficiency competent e coli  (New England Biolabs)

Journal: bioRxiv

Article Title: Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression

doi: 10.64898/2026.01.27.701873

Figure Lengend Snippet: ( A ) Homing restriction–religation (HRR) workflow for reconstituting plasmids from RCA concatemers maintains the functionality of the original vectors. ( B, upper ) Gel electrophoresis of DNFs before and after addition of increasing concentrations of EDTA. Both the soluble fraction and the pellet (after centrifugation) are shown. EDTA treatment shifts DNF material from the pellet into the soluble fraction, indicating solubilization. ( B, lower ) Photographs of the tube bottoms after centrifugation of DNFs, showing that EDTA removes the visible pellet. ( C ) RCA performed with decreasing amounts of solubilized DNF as template. Robust amplification is observed down to ∼200 DNFs, indicating solid amplification sensitivity. ( D ) Restriction analysis of the vector used to develop HRR (pMC174). Digestion with I-CeuI, I-SceI, or PI-SceI converts the original concatemer (C) into linear plasmid species (M) and ligated. ( E ) Plates showing colonies from transformation of 10β bacterial cells with pMC174 either digested with the homing endonuclease pool or left undigested. Colony formation occurs almost exclusively after digestion with the enzyme pool, consistent with reconstitution of viable, monomeric plasmids following cleavage and ligation. ( F ) Agarose gel showing original and reconstituted (R) plasmids from a metagenomic library and a GFP-expressing vector; both pairs run at comparable sizes and display similar migration patterns. ( G ) Functional copy number measured after transformation of either the original or reconstituted GFP vector into an expression strain of E. coli; reconstituted plasmids show a 10–20% decrease in functional copies. ( H ) PFAM domain distribution in the metagenomic library before and after plasmid reconstitution by PCR or HRR; HRR-reconstituted plasmids retain domain frequencies comparable to the original library, whereas PCR introduces severe shifts. For the analysis, the 100 most abundant PFAM annotations were considered.

Article Snippet: For protein expression, NEB ® T7 Express High Efficiency Competent E. coli (C2566) was used. pET-T7-sfGFP, pET-T7-mCherry, pUCT7-sfGFP, JJB668, JJB270, pMC174, pMiniT2.0 (included in NEB’s cloning kit # E1202) were made using NEBuilder HiFi DNA Assembly Cloning Kit (NEB), and gene/DNA oligonucleotide synthesis was provided by IDT (Coralville, IA).

Techniques: Nucleic Acid Electrophoresis, Centrifugation, Amplification, Plasmid Preparation, Transformation Assay, Ligation, Agarose Gel Electrophoresis, Expressing, Migration, Functional Assay

Journal: bioRxiv

Article Title: Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression

doi: 10.64898/2026.01.27.701873

Figure Lengend Snippet: (A) A 15-nucleotide single-stranded DNA (ssDNA) substrate labeled with a 5′ fluorescein and a 3′ quencher serves as a reporter for ssDNA-binding proteins. Upon protein binding, the substrate transitions from a quenched, intramolecularly folded conformation to an extended state that spatially separates the fluorophore and quencher, thereby restoring fluorescein fluorescence. (B) A genomic library from E. coli was constructed by mechanical fragmentation and cloning of 6–10 kb inserts into the pMC174 backbone (EV, empty vector). (C) Representative FADS sorting step showing the gating of IVTT droplets generated from DNF derived from the E. coli library and supplemented with the 15-nt ssDNA substrate. Microdroplets exhibiting fluorescence levels approximately threefold above the main population were selected, corresponding to ∼0.05% in round 1 and ∼0.1% in round 2. Forward scatter (FSC) reflects microdroplet size. (D) Enrichment of RecA Pfam Annotations During Screening. A progressive increase in RecA Pfam annotations per contig was observed, rising from an initial 2% to 229% after two rounds of selection. (E) Validation of RecA ssDNA-binding activity. Binding of RecA to the 15-nt oligonucleotide results in a gradual increase in fluorescence.

Article Snippet: For protein expression, NEB ® T7 Express High Efficiency Competent E. coli (C2566) was used. pET-T7-sfGFP, pET-T7-mCherry, pUCT7-sfGFP, JJB668, JJB270, pMC174, pMiniT2.0 (included in NEB’s cloning kit # E1202) were made using NEBuilder HiFi DNA Assembly Cloning Kit (NEB), and gene/DNA oligonucleotide synthesis was provided by IDT (Coralville, IA).

Techniques: Labeling, Binding Assay, Protein Binding, Fluorescence, Construct, Cloning, Plasmid Preparation, Generated, Derivative Assay, Selection, Biomarker Discovery, Activity Assay