dh5α chemically competent cell  (New England Biolabs)

Journal: bioRxiv

Article Title: GRASP: a modular toolkit for building synthetic pentatricopeptide repeat RNA-binding proteins

doi: 10.1101/2025.06.29.661641

Figure Lengend Snippet: Reconstruction and functional assay of 9SrpoADYW. ( A ) The TRX-9S-DYW synthetic PPR protein was reconstructed using GRASP modules, with an N-terminal thioredoxin solubility tag, and a C-terminal consensus RNA editing domain . We named this iteration 9SrpoADYW. The rpoA binding and editing site is positioned after the 9SrpoADYW stop codon, so 9SrpoADYW edits the same transcript it is translated from. ( B ) SDS-PAGE showing expression of 9SrpoADYW in an E. coli cell-free expression reaction and Western blot showing 9SrpoADYW detected using anti-His tag antibody. Full acrylamide gel and western blot images are provided in Supplementary Fig. S3. ( C ) Sanger chromatograms of cDNA amplified from E. coli cell-free lysate expression reactions shows that 9SrpoADYW assembled with the GRASP kit is functional. Sanger chromatogram peak proportions were determined using MultiEditR .

Article Snippet: 3μL of the assembly mix was transformed into chemically competent DH5α E. coli cells (ThermoFisher, Waltham, Massachusetts, USA).

Techniques: Functional Assay, Solubility, Binding Assay, SDS Page, Expressing, Western Blot, Acrylamide Gel Assay, Amplification

Journal: bioRxiv

Article Title: GRASP: a modular toolkit for building synthetic pentatricopeptide repeat RNA-binding proteins

doi: 10.1101/2025.06.29.661641

Figure Lengend Snippet: Heatmaps displaying editing of RNA targets catalysed by 9SrpoADYW and its variants, and model coefficients from a logistic regression analysis of the RNA editing data. In all cases the reference is the editing seen with the unmodified protein (p0) and/or the unmodified target (r0). ( A ) 9SrpoADYW (p0) and 30 modified variants (p1-p30) were expressed in E. coli cell-free expression lysate and incubated with a pool of in vitro transcribed RNA targets comprising rpoA (r0) and single nucleotide sequence variants (r1-45). The values indicate the mean (n = 3) proportion of edited reads; ( B ) Protein:RNA interaction coefficients indicating ‘specificity’; ( C ) Maximum pooled interaction coefficients for cognate interactions between p1:r1 to p30:r30 shows the base preference for each dsnSc motif variant; ( D ) Maximum pooled interaction coefficients preceding and following cognate motif/base interactions.

Article Snippet: 3μL of the assembly mix was transformed into chemically competent DH5α E. coli cells (ThermoFisher, Waltham, Massachusetts, USA).

Techniques: Modification, Expressing, Incubation, In Vitro, Sequencing, Variant Assay