## Results: 4

Figure 4. From: A stochastic single-molecule event triggers phenotype switching of a bacterial cell.

Figure 1. From: A stochastic single-molecule event triggers phenotype switching of a bacterial cell.

*E. coli.*(A) The repressor LacI and permease LacY form a positive feedback loop. Expression of permease increases the intracellular concentration of the inducer, TMG, which causes dissociation of LacI from the promoter, leading to even more expression of permeases. Cells with a sufficient number of permeases will quickly reach a state of full induction, while cells with too few permeases will stay uninduced. (B) After 24 hours in M9 media containing 30 µM TMG, strain SX700 expressing a LacY-YFP fusion exhibits all-or-none fluorescence in a fluorescence-phase contrast overlay. Fluorescence imaging with high sensitivity reveals single molecules of permease in the uninduced cells zoomed in on the red box from B. (C) After one day of continuous growth in media containing 0 to 50 µM TMG, the resulting bimodal fluorescence distributions show that a fraction of the population exists either in an uninduced or induced state, with the relative fractions depending on the TMG concentration. (D) The distributions of LacY-YFP molecules in the uninduced fraction of the bimodal population at different TMG concentrations, measured with single-molecule sensitivity, indicate that one permease molecule is not enough to induce the

*lac*operon, as previously hypothesized (12). Over 100 cells were analyzed at each concentration. Error bars are standard errors determined by bootstrapping.

Figure 2. From: A stochastic single-molecule event triggers phenotype switching of a bacterial cell.

*p*, is fit with a Hill equation

*p*=

*y*

^{4.5}/ (

*y*

^{4.5}+ 375

^{4.5}) for initial permease number,

*y*. The threshold of permease numbers for induction is thus determined to be 375 molecules. Error bars are the inverse square root of sample size at each point. (C) To prove that complete dissociation of tetrameric repressor from two operators triggers induction, we constructed strain SX702 with auxiliary operators removed (no DNA looping). The figure shows single-cell traces of permease numbers in single cells grown in 40 µM TMG as a function of time. Unlike the looping strain SX700, the rapid induction of SX702 is no longer dependent on the initial number of permease molecules. This proves that phenotype switching is the result of a complete dissociation of the tetrameric repressor as shown in B. (D) In the absence of DNA looping, the entire population of strain SX702 rapidly induces in a coordinated manner from far below the threshold for a concentration as low as 20 µM TMG. DNA looping is necessary for bistability of the

*lac*operon under these conditions.

Figure 3. From: A stochastic single-molecule event triggers phenotype switching of a bacterial cell.

^{2}/σ

^{2}(purple circles ) and σ

^{2}/µ (gray triangles) suggest that the frequency of large bursts is independent of the TMG concentration but that the burst size is not.