TetR tetO2 variant repression landscape by in vivo sort-seq
The highly rugged yet navigable regulatory landscape of the bacterial transcription factor TetRAn eight-position combinatorial tetO2 transcription-factor-binding-site library (4^8 = 65,536 theoretical variants) was cloned into a low-copy plasmid reporter in Escherichia coli. TetR-mediated repression was measured by GFP fluorescence after FACS sorting into 13 bins and amplicon sequencing; the processed table contains the 17,765 variants in the QC-filtered giant component used for landscape analyses.
Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.
Perturbation & assay details
Basal / No anhydrotetracycline (Atc)
Plasmid-based bacterial reporter using the low-copy pBBR1 origin, constitutive TetR expression from a weak pLac promoter variant, and sfGFP driven by BBa_J23110 with the tetO2 variant placed at the +10 position relative to the transcription start site. A RiboJ insulator separates TFBS sequence effects from the reporter 5′ UTR. Cells were sorted into 13 fluorescence bins and the TFBS amplicons were sequenced; lower GFP fluorescence indicates stronger repression. The library exhaustively randomizes positions 5–8 and 12–15 of the 19-bp tetO2 sequence.
Processed data
50 rows per page. Click a cell to inspect its full value.
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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.
Column dictionary · 8 definitions
- variant_id
- Stable package identifier formed as tetO2_<sequence>.
- sequence
- 19-bp DNA sequence of the tested tetO2 transcription-factor-binding site variant.
- distance_to_wild_type
- Hamming distance from wild-type tetO2 across the eight randomized positions (positions 5–8 and 12–15).
- normalized_repression
- Article-provided repression strength normalized to wild-type tetO2; 1 corresponds to the wild-type reference and larger values indicate stronger repression.
- landscape_class
- Peak if all observed single-mutant neighbors have lower repression; Other for non-peak variants.
- peak_type
- High for peaks above wild-type repression, Low for other peaks, and blank for non-peak variants.
- basin_size
- Number of variants from which an accessible increasing-repression path to the peak exists; blank for non-peak variants.
- relative_connectivity
- Observed fraction of the 24 possible single-nucleotide neighbors represented in the empirical genotype network.
Quality control
The publication trimmed adapters, merged paired reads, retained high-quality sequences at Q ≥ 33, and removed sequences with mutations or indels outside the variable TFBS library region. It then removed variants absent from any of the three replicates, required at least 30 reads across the 13 bins, calculated replicate repression estimates, and excluded variants with repression coefficient of variation above 0.5. The processed table further retains only the 17,765-variant largest weakly connected component used for the paper's landscape analyses and validates unique 19-bp A/C/G/T sequences with finite repression and connectivity values.
Curation notes
The processed table was generated from the article source workbook sheet 'Figures 3a-d; S14-S16' and peak basin annotations from 'Figures 4; S17'. This is the canonical 17,765-variant landscape table used in the paper. The workbook's separate 'Figures 2b; 2d; S11' sheet contains 19,370 rows with replicate count summaries and normalized values that do not exactly match the landscape sheet; it is retained in raw_data but was not mixed into the canonical effect scores. The wild-type tetO2 reference sequence is TCCCTATCAGTGATAGAGA and serves as the normalization reference, although it is not a row in the 17,765-variant landscape table.