Journal: bioRxiv
Article Title: Mobile DNA is replete with hotspots for the de novo emergence of gene regulation
doi: 10.1101/2023.10.22.563463
Figure Lengend Snippet: (a) We pooled parental IS fragments for error-prone PCR, (b) cloned the mutagenized library into the dual reporter plasmid pMR1, and transformed the resulting plasmid library into E. coli . Inserts with promoter activity fluoresce green or red (shown as blue or red here), depending on the orientation of the newly created promoter, and with different intensities based on the promoter strength. (c) We sorted bacteria using fluorescence activated cell sorting (FACS) into four bins for each fluorescence color, corresponding to none, low, medium, and high fluorescence for both GFP and RFP (thus 8 bins total). We isolated inserts from cells in each bin and sequenced them using Illumina sequencing. (d) Percentages at the top of the figure: for each parent sequence, the probability of a mutation creating an active promoter de novo (P new ). For each parent sequence (x-axis), the height of the vertical bars shows the percent of mutations creating promoters with expression strengths in each of four color-coded categories (color legend, blue: GFP, red: RFP). Note: the y-axis begins at 70%. (e) The probability of a mutation creating an active promoter de novo in the parent sequences (P new ) for both the top strand (blue: GFP) and bottom strand (red: RFP). (f) Percent of de novo promoters in each strength category (white to blue, see color legend) based on the number of mutations. Note: the y-axis begins at 80%. (g,h) Single mutations observed for each parental IS fragment (rows) and each nucleotide position (columns), together with the new gene expression they drive (blue or red, see color legend). Gray boxes indicate that no mutagenized fragment harbors the indicated nucleotide. Boxes with black circles indicate the wild-type sequence. Sequences are shown from the 5’ to the 3’ end. (g) Expression level of top DNA strand (blue, darker: higher expression). (h) Expression level of bottom DNA strand (red, darker: higher expression).
Article Snippet: We immediately transformed the cloned products into E. coli DH5α electrocompetent cells (Takara, Japan, product #9027), adding 2 uL of the product to 100 uL of electrocompetent cells.
Techniques: Clone Assay, Plasmid Preparation, Transformation Assay, Activity Assay, Bacteria, Fluorescence, FACS, Isolation, Sequencing, Mutagenesis, Expressing
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