Relative Promoter-Strength Characterization of a Synthetic Anderson Library in E. coli via Flow-Cytometric sfGFP Reporter Quantification (RPU Normalization)

Each promoter-reporter strain assayed in 3 biological replicates (separate colonies) across 2 independent days, with 4 technical reads each. Cultures harvested at standardized mid-exponential phase (OD600 0.3-0.5). A blank (no-promoter/promoterless sfGFP) and the RPU reference (J23101) are included on every plate and run. Well/sample order randomized; cytometer voltages fixed across all samples within a session.

BSL-1 (non-pathogenic E. coli DH10B). PPE: lab coat, gloves, goggles. Flow cytometer sheath/waste handled per instrument SOP; treat cell waste with 10% bleach before disposal. Kanamycin is an irritant. No high-power lasers exposed to user (enclosed cytometer). Autoclave plasticware and culture waste; decontaminate spills with 70% ethanol.

Positive control: BBa_J23101 reference standard (defines 1 RPU) on every run. Negative control: promoterless sfGFP construct measures autofluorescence and transcriptional leak, subtracted from all values. Untransformed DH10B sets the true autofluorescence floor. Bead control: 8-peak calibration beads verify instrument stability and enable MEFL conversion. Medium-only blank checks contamination.

Promoters span ~0.05 to ~3 RPU. Unimodal, log-normal single-cell distributions (CV typically 30-60% biological noise) with clear separation between strong and weak promoters. Reference J23101 = 1.00 RPU by definition. Day-to-day RPU CV <15% for a given promoter indicates good normalization. Promoterless control should be near autofluorescence floor.

• E. coli DH10B harboring promoter-sfGFP plasmids (pSB3K3 or similar, kanamycin)
• Reference standard strain: BBa_J23101-RBS-sfGFP (defines 1 RPU)
• Promoterless sfGFP construct (autofluorescence/leak control)
• Supplemented M9 minimal medium: M9 salts, 0.4% glycerol, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2, thiamine
• Kanamycin, 50 ug/mL
• PBS pH 7.4 for dilution
• Flow cytometry sheath fluid; rainbow calibration beads (8-peak)
• 96-well deep-well plates and U-bottom read plates

To quantify the relative strength of a set of synthetic constitutive promoters (e.g., Anderson collection variants) driving sfGFP in E. coli DH10B by single-cell flow cytometry, converting raw fluorescence to relative promoter units (RPU) against the BBa_J23101 reference standard for portable, lab-independent values.

1. Inoculate each strain from a single colony into 0.5 mL supplemented M9 + kanamycin in a deep-well plate; grow overnight at 37 C, 1000 rpm shaking.
2. Dilute 1:100 into fresh medium; grow ~4-6 h to OD600 0.3-0.5 (mid-exponential).
3. Run 8-peak calibration beads to set/verify cytometer voltages; keep gains fixed all session.
4. Dilute cultures 1:100 in cold PBS to ~10^6 cells/mL.
5. Acquire >=50,000 events per sample on FITC channel (ex 488/em 530); gate on FSC/SSC to exclude debris and doublets.
6. Acquire the promoterless control and J23101 reference identically each run.
7. Compute per-sample geometric mean fluorescence after gating.
8. Repeat across 3 colonies and 2 days.
9. Convert to RPU: RPU = (meanpromoter - meanblank)/(meanJ23101 - meanblank).

Independent: promoter identity/sequence. Dependent: per-cell sfGFP geometric mean fluorescence and derived RPU. Controlled: host strain (DH10B), plasmid backbone/copy number, RBS, growth phase (OD600 0.3-0.5), medium, temperature 37 C, cytometer voltages/gains, antibiotic.

Within an isogenic background and standardized growth state, per-cell sfGFP fluorescence scales linearly with promoter transcriptional output, allowing promoters to be rank-ordered over ~2 orders of magnitude. RPU-normalized values will be reproducible across days within 15% CV, removing instrument-gain dependence.

Process FCS files in FlowJo or Python (FlowCytometryTools/flowkit). Gate FSC/SSC singlets, compute geometric mean FITC. Subtract blank, divide by reference to get RPU. Optionally convert arbitrary units to MEFL via bead standard curve. Aggregate replicates; report mean RPU +/- SD and rank-order promoters. Plot distributions and a strength ladder.

1. Bimodal distribution: plasmid instability or mixed population — re-streak single colonies and confirm antibiotic selection. 2. RPU drifts between days: growth phase not standardized — strictly harvest at OD600 0.3-0.5 and re-run beads. 3. Strong promoter saturating detector: reduce PMT voltage and re-acquire all samples at the new setting. 4. Weak promoter indistinguishable from blank: subtract autofluorescence carefully and increase event count to 100,000. 5. High debris: improve FSC/SSC gating and filter PBS.

Compare promoter RPUs by one-way ANOVA on log-RPU with Tukey HSD across all promoters; alpha=0.05. Day and colony entered as random effects in a mixed model to confirm normalization removes batch variance. Power: with n=6 (3 colonies x 2 days), detecting a 1.5-fold RPU difference (log SD ~0.1) achieves >90% power at alpha=0.05. Report 95% CIs on RPU.