Fig. S4.

Estimation of absolute transposase numbers from Venus-TnpA bleaching kinetics. (A) Individual bleaching curves are exponential. Bleaching curves of normalized Venus-TnpA fluorescence vs. time in units of each curve’s fitted decay constant from 419 individual cells (blue points) compared with exponential bleaching kinetics (black line). (Inset) A representative single cell bleaching trace over 10 s. We truncate the bleaching curve (e.g., at vertical dashed line) when the fluorescence comes within 100 AU of the background (horizontal dashed line). (B) Individual bleaching traces (blue points) with 100 ms exposure time compared with the mean bleaching kinetics (black line). The mean decay constant is τ = 0.68 ± 0.01 s. Fluctuations from the mean bleaching kinetics are binomial noise, and analysis of the variance allows estimation of absolute Venus-TnpA numbers. (C) Mean normalized variance from 419 cells (black points) vs. fraction of bleached molecules, (1 – p). The proportionality constant, ν, relating fluorescence to number of molecules is related to the area under the best fit parabola [red line], yielding ν = 130 ± 10.