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. 2020 Jul 1;48(14):8165–8177. doi: 10.1093/nar/gkaa557

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — Inducible sgRNA response times are consistent with the free-running oscillator. A single constitutive promoter (PAx) of the free-running system was replaced with a PhlF repressor-based promoter system, allowing inducible control with small molecule DAPG. Measurement of cells in the mother machine allows explicit tracking of individual cell responses to added inducers to determine characteristic times Δt of sgRNA repression cascade steps. Escherichia coli MG1655 cells with plasmids were grown in EZ-RDM. (A) One-step sgRNA repression is measured. Expected behavior is YFP bright to dim upon DAPG induction (A4NT sgRNA). Cells are re-entering exponential growth and equilibrating prior to aTc induction. Plot shows 599 cell reporter traces of plasmid version (pJK429). 1 frame = 6 min. (B) Two-step sgRNA repression is measured. Expected behavior is YFP dim to bright upon DAPG induction (A2NT sgRNA). (The initial 1/0 YFP accounts for repression from leaky dCas9 expression.) Plot shows 515 cell traces of plasmid version (pJK430). (C) Histograms of measured characteristic time steps indicated in (A) and (B). Percentages are percent repression or de-repression. Distributions were measured from (A) 2819 cells for pJK429 and (B) 3136 cells for pJK430.