Fig. 1.
Gene expression noise drug screening of a minimal HIV gene circuit using time-lapse fluorescence microscopy. Clonal samples of Jurkat T cells infected with a minimal HIV feedback circuit LTR-d2GFP-IRES-Tat (Ld2GIT), treated with 1,806 unique compounds at t = 0, were prepared and imaged for 48 h. LTR: HIV 5′ long terminal repeat promoter; d2GFP: destabilized green fluorescent protein, half-life = 2.55 h; IRES: internal ribosome entry site; Tat: HIV Tat protein, provides positive feedback and enhances HIV transcription from the LTR promoter. (A) A sample of noise processing from plate 2 well K16. Eighteen-hour single-cell time-dependent d2GFP fluorescence intensities are extracted from the raw images of a clonal cell population, separate for each drug treatment, and are processed for gene expression noise and noise autocorrelation trajectories (1, 22). (B) Representation of a noise-space coordinate. For each drug treatment, a noise-space coordinate is made up of three parameters: mean fluorescence (<FL>), high-frequency noise magnitude (HF-CV2), and high-frequency noise half autocorrelation time (HF-τ1/2) (28); 1,806 noise-space coordinates for all screened compounds are displayed in (C) HF-CV2 – <FL> representation and (D) HF-τ1/2 – <FL> representation. TNF-α acts as positive control, while Alp acts as negative control. Drugs that significantly modulate HF-CV2 or HF-τ1/2, beyond 2 SDs from daily untreated average, are colored red according to a daily gating strategy. Solid lines represent models calculated from two experiments on a nonfeedback polyclonal Jurkat cell line infected with an LTR-d2GFP (Ld2G) gene circuit, which is a nonfeedback version of Ld2GIT. Compounds show a large range of noise modulation in both CV2 and τ1/2 space. Error bars represent one SD from the mean untreated values (black squares).