Supplementary Materials

Stochastic antagonism between two proteins governs a bacterial cell fate switch

Nathan D. Lord, Thomas M. Norman, Ruoshi Yuan, Somenath Bakshi, Richard Losick, Johan Paulsson

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Mathematical Derivations
  • Figs. S1 to S21
  • Captions for Movies S1 to S4
  • References

Images, Video, and Other Media

Movie S1
Switching driven by stochastic antagonism in a reduced circuit in Bacillus subtilis. The movie shows cells of a B. subtilis strain (TMN1159) as they grow in microfluidic channels. These cells leave SinR under its native constitutive promoter, but replace SinI with an IPTG-inducible promoter to bypass any endogenous transcriptional regulation. Cells also contain a PtapA-mKate2 fluorescent reporter, which is strongly responsive to SinR abundance. The movie shows a series of experiments side-by-side in which bacteria were grown in the presence of constant amounts of inducer as indicated by the labels. As IPTG concentration is raised, SinI levels increase relative to the constant amount of SinR, and reporter pulsing increases in frequency. Note that the channels are constantly loaded with cells throughout the movie, as confirmed by a constitutively expressed segmentation marker (not shown).
Movie S2
Switching driven by stochastic antagonism reconstituted in Escherichia coli. The movie shows cells of an E. coli strain (NDL-423) as they grow in microfluidic channels. These cells are engineered to contain SinR under a constitutive promoter and SinI under an IPTG-inducible promoter. Cells also contain a synthetic SinR-repressed GFP reporter (left panel), which can be used to monitor the level of SinR activity, and a constitutively expressed segmentation marker (right panel), which can be used to verify constant loading of the channels with cells throughout the movie. The rows in the panels show a stacked series of experiments in which bacteria were grown in the presence of constant amounts of inducer as indicated by the labels. As IPTG concentration is raised, SinI levels increase relative to the constant amount of SinR, and reporter pulsing increases in intensity.
Movie S3
Effect of Slr on switching in E. coli reconstitution. The movie compares switching in the E. coli reconstitution strain with (NDL-419, '+Slr' in movie) and without (NDL-423, '-Slr' in movie) a synthetic SinR-repressed copy of slr. In both strains SinR production is constitutive, while SinI production is controlled by an IPTG-inducible promoter. Levels of inducer (60 μM for NDL-419 and 100 μM for NDL- 423) were chosen to approximately match the initiation frequency of switching between the two strains, so that differences in duration are more apparent. Note the one long-lived event in the '-Slr' lane is a dying cell that has filamented.
Movie S4
Effect of clpXP mutation on switching in the E. coli reconstitution. For technical reasons, clpXP mutations were introduced into the E. coli reconstitution strains when engineering Slr feedback (see details in the strain construction section of Materials and Methods). The movie shows switching in the strain TMN1221, which is identical to the NDL-423 strain used in the rest of the paper but with clpXP intact, in response to varying concentrations of IPTG as indicated by the label. The left column shows the synthetic SinR-repressed reporter used throughout the paper to monitor SinR activity, and the right column shows a constitutive segmentation marker used to track cell position within the microfluidic device.