Journal: bioRxiv

Article Title: The displacement of the σ 70 finger in initial transcription is highly heterogeneous and promoter-dependent

doi: 10.1101/2023.06.10.544452

Figure Lengend Snippet: An smFRET assay for detecting σ-finger movements in solution. (A) Conformational state of the pre-displaced σ-finger in E. coli RP itc4 (PDB 4YLN). Grey, black and blue ribbons show the template DNA strand, non-template DNA strand and transcribed RNA, respectively. Straw ribbons and mesh show σ 70 . (B) smFRET labelling scheme. Orange and green spheres: fluorescent probes at residues 511 and 366 of σ 70 . Purple sphere: catalytic Mg 2+ ion. (C) Schematic of the assay used for experiments tracking σ-finger movements. Colour scheme as in panel (A) but with the addition of RNAP core shown in light grey. (D) lacCONS DNA fragment used for the assay to track σ-finger movements at RP itc2 . (E) smFRET data for the σ-finger in RP itc≤2 complexes formed using ApA dinucleotide primer showing static ( upper ) and dynamic ( lower ) behaviour. Left, representative traces of static and dynamic behaviour. Right, E* histograms formed as a result of hidden Markov modelling, and Gaussian fitting of sub-populations.

Article Snippet: Hexahistidine-tagged Escherichia coli RNAP core enzyme was prepared using co-expressed genes encoding RNAP β’, β, α, and ω subunits to afford an RNAP core enzyme as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pEcABC-His 6 and plasmid pCDFω , were used to inoculate 20 ml LB broth containing 100 μg/ml ampicillin, and 50 μg/ml kanamycin.

Techniques: Smfret Assay

Journal: Genome Biology

Article Title: Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants

doi: 10.1186/s13059-023-02929-6

Figure Lengend Snippet: High-throughput characterization of DNA cleavage activities of LbCas12a point mutations in E. coli . A Schematic representation of bacterium-based selection assay to isolate LbCas12a mutants with enhanced activity. E.coli BW25141:DE3 cells containing an inducible ccdB expression plasmid were transformed with LbCas12a variant library, which was programmed to cleave the reporter plasmid through a target site with TTTT PAM sequence. Active LbCas12a mutants with enhanced activity enable the clearance of reporter plasmid, thus avoiding the cell death upon the induction of ccdB expression with arabinose. LbCas12a plasmids from survived cells were extracted and used for subsequent round of selection. Four rounds of sequential selection were performed. Round 3 and 4 libraries were sequenced and used to calculate the enrichment of LbCas12a variants. B Design of a saturation mutagenesis library for LbCas12a. Every codon of LbCas12a was randomized with NNK degenerate primers by nicking mutagenesis . Importantly, each member of plasmid library only contains one codon change at a time, which was verified by Sanger sequencing of 24 individual plasmids from the library. C Enrichment scores for ~ 9000 LbCas12a point mutations over the last round of selection (round 4). Synonymous variants without changes on the protein sequences were colored in red. Variants with a minimal of 50 counts were included in this analysis. D Enrichment scores for 5 selected positions of LbCas12a that have been evaluated prior to this study

Article Snippet: Transformed E. coli BL21 (DE3) cells (EMD Millipore) were grown in TB medium with 50 μg/mL Kanamycin until OD 600 reaches 0.6–0.8.

Techniques: High Throughput Screening Assay, Selection, Activity Assay, Expressing, Plasmid Preparation, Transformation Assay, Variant Assay, Sequencing, Mutagenesis