Peer review of: Exploiting natural chemical photosensitivity of anhydrotetracycline and tetracycline for dynamic a (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Summary 
This manuscript introduced a novel dynamic cellular control approach utilizing the photosensitivity 
of aTc and Tc, with easy control over active molecules level using UVA light, accompanied with a 
mathematical model of the light dependency. In addition to describing this new regulator system, 
the author also showcased its application in control of gene expression, growth and reducing dark 
state activity, and improved the effective range of aTc through the construction of the chemical 
bandpass filter. This method could potentially be a valuable tool in optogenetic induction 
experiments and expand the focus of photosensitive molecules. The manuscript in general is well 
written with comprehensive analysis, but a few issues still need to be clarified/resolved. 
 
Comments: 
1. The authors showed that effective inactivation of aTc was achieved using UVA light - what about 
the phototoxic effect of UVA light on the other components of the experiments beside aTc(e.g. 
damage to other cellular macromolecules)? Does this limit the usage of this control system in 
certain experiments? 
 
2. For all the figure captions, I suggest the authors break down and clearly list the captions of 
each panel, instead of lumping all the description in one paragraph in the caption. For example, I 
do not find a detailed description of 1D. 
 
3. While the mathematical models can predict the aTc degradation separately in TetR and rTetR 
systems, can such prediction still be achieved in the concentration bandpass filter system to 
enable controllability of aTC inactivation? 
 
4. While the majority of the analysis were focused on aTc, the last section on growth control 
utilized Tc instead, which makes the reader wonder -does Tc and aTc respond to UVA light 
similarly? Further discussion/data could facilitate future users of this system better design their 
experiments 
 
5. In the same section discussion growth control using Tc, the statement “ 2) that increasing Tc 
concentrations result in increasing growth rates” does not seem right. Did you mean increasing 
UVA exposure time instead of Tc concentration? 
 
6. The figures were discussed out of order - Fig 4 was mentioned before Fig 3C and 3D. Consider 
reorganize the figures/or result sections. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In their manuscript entitle “aTc and Tc as light-sensitive controllers – exploiting natural chemical 
photosensitivity for dynamic optogenetic set point control”, Baumschlager and colleagues describe 
an often-overlooked property of bioactive small molecules: their photosensitivity. By degrading 
anhydrotetracycline with pulses of UV-A light, they show that the activation of a Tet-responsive 
promoter can be toggled from on to off or set to an intermediate level. 
 
I have a very high opinion of the authors’ optogenetic work to date, as well as their creativity in 
applying an industrial detoxification approach as a cell biology tool. However, I am a little doubtful 
about the possible utility of photo-degradation of aTc as an “optogenetic” approach. 
 
This skepticism comes from a few sources: (1) that light-based control only goes 1 way (you 
cannot reverse the process and recover functional aTc), (2) photodegradation and UV-A light can 
both be potent sources of toxicity for cells, and (3) Tet-responsive promoters can be tuned even 
more easily by simply varying the concentration of aTc applied to a population of cells, without the 
need to calibrate light intensity to the geometry of a particular system, the cell density and 
absorbance of the media, etc. 
 
The authors may argue against (3) that their system enables acute removal of aTc, which is 
difficult to do otherwise (at least without a spin-down & resuspension in fresh media), but as they 
point out, we already have TetR and rTetR, so you can always use the ‘reverse’ repressor system 
to flip the sign of aTc addition (so that acute removal is replaced with acute addition, something 
that is very easy to achieve). 
 
In summary: while the work is technically sound, my skepticism about the system’s overall utility 
leads me to believe that the work does not rise to the level of general interest of Nat Comms & 
may be better suited for a more specialized synthetic biology journal. 
 
Beyond these high-level concerns, I have some specific concerns regarding the authors’ 
characterization of photo-degradation of aTc: 
 
(1) Dense bacterial cultures tend to absorb visible light – this is the basis for the popular “optical 
density” measurements of cell density in liquid culture. Although OD at 600 nm is usually used to 
characterize density, liquid cultures are strong absorbers at shorter wavelengths, raising the 
possibility that all of the authors’ measurements of light-to-aTc degradation are extremely cell 
density dependent (otherwise aTc in the middle of the culture will be hit by a lower light dose and 
thus protected from degradation). 
 
(2) What is the chemical structure of the degradation product? This is actually a rather important 
consideration, because completely photoconverting the aTc in solution will lead to high 
concentrations of its photo-degradation products. We must be sure that these are not themselves 
biologically active, and it is worth addressing whether free radicals induced during photo-
degradation might impair other cellular functions or invoke a stress response. 
 
(3) Can the authors perform any studies to test for deleterious effects of UV-A radiation on cellular 
function? In addition to the possibility of degradation products negatively affecting cellular 
function, light in the 300-400 nm range is almost always accompanied by phototoxicity or DNA 
damage. It could be the case that the doses used here are too low to induce a large-scale cell 
stress response, but this must be validated with data (a UV dose response). Ideally, the authors 
could obtain UV dose responses to cell growth rate and stress pathway activation, both in the 
presence and absence of aTc.