Design and benchmarking
of a noise tuning system. (a) Schematic
depiction of the noise tuner. In a Tet-ON system21 doxycycline activates transcription of the fluorescent
mNeongreen reporter gene by binding to the constitutively expressed
reverse tetracycline trans-activator (rtTA, not shown). Addition of
theophylline prevents mRNA degradation by disrupting the ribozyme
cleavage activity in the 3′-UTR. (b) Comparison of the noise
tuner with constructs harboring native 3′ regulatory regions
measured by flow cytometry. All constructs are identical in their
Tet promoter and mNeongreen fluorescent reporter gene and differ only
in their 3′ RRs that confer low (FZF1t, GIC1t) or high (TPS1t,
ADH1t) expression.24 Top panel: Dose–responses
with 0 to 12.8 ng/μL doxycycline. The noise-tuner transcript
was either unstable (0 mM theophylline, “NT”) or fully
stabilized (12.8 mM theophylline, “NT + theo”). Bottom
panel: The noise tuner exhibits similar inverse noise-median correlation
as native 3′ RRs. Noise is given as the robust coefficient
of variation (robust CV, see Methods). Median
fluorescence intensities normalized to a constitutively expressed
mTurquoise2 reporter gene are given in arbitrary units (a.u.). Higher
median fluorescence intensities correspond to induction with higher
doxycycline concentrations.