Peer review of: De novo design of an intercellular signaling toolbox for multi-channel cell–cell communication and (2020)

Reviewers' Comments:



Reviewer #1:
Remarks to the Author:


Factual summary
Six novel orthogonal cell-cell signaling channels were ported to E. coli from diverse organisms such as Pseudomonas sp., Yersinia sp., Streptomyces sp., Rhodobacter sp. and others. Synthesis pathways for the molecules and genetic sensor components were developed and characterized in E. coli for use in synthetic biology. The sensor components of two signaling modules were further optimized through directed evolution. The signaling modules were then successfully ported to mammalian HEK-293T cells and yeast S. cerevisiae and used for intra- and inter-kingdom communication. Finally, the communications modules were used to construct simple devices in E. coli, including a spatial pattern formation system and a spatially distributed biological logic gate system.

Strengths and weaknesses
Some of the much-needed cell-to-cell communication tools for use in Synthetic Biology are developed in this study. Not only are these tools implemented for use in E. coli; some are shown to be functional in yeast and mammalian cells and could, thus, be used for inter- kingdom communication. The study also implements original and exciting spatially segregated logic gate circuitry that has the potential to be of good interest to the Synthetic Biology community.
The developed signaling tools have not been assessed for their effects on E. coli growth and a metabolic burden analysis is absent. This should be included in the final manuscript. A further weakness is linked to the number of communication channels implemented in organisms other than E. coli: only a single communication channel has been implemented in mammalian cells, whereas two have been ported to S. cerevisiae. This raises some doubt as to the potential for the portability of the other channels. Finally, the sequences of the developed components are not clearly presented in the Supplementary information; a comprehensive list of all used and developed genetic tools has to be included. Important: the genetic sequences have to be in copiable text format.

Verdict
I endorse the publication of this paper after the following revisions have been implemented:
1. Please provide a complete and comprehensive cell burden analysis; how constructs
impact growth in E. coli (i.e. provide growth curves).
2. Detailed presentation of all genetic sequences and constructs.
3. Minor, but potentially useful: include characterization of signaling modules in
different growth media in E. coli (M9, LB, 2xYT).
4. Expand referencing in Discussion.
5. Include plots of actual values presented on the heatmaps.
6. Native English copy-editing

Comments
• Minor language corrections throughout; only a small subset of what has to be done is mentioned here. In general it is OK, but lacks the native English form.
    o Figure 1 legend sounds strange in past tense.
    o pg7, lines 244-onwards; a little messy English. Replace ‘induction-fold’. o pg8, line 288; ‘model cells’ → ‘cell models’
    o pg8, line 299; it is not clear what you mean by ‘basal promoter’
    o pg25, line 757; replace ‘shots’ ; pg11, line 409 : "mTurquoise2"

• Results
o pg5, lines 151-154: include more detailed description of how promoters were
designed, where the parts came from.
o Include some measure of burden that the sensors presented in this study
have on host cells; this can be growth of cells with fully active signaling and sensing modules; alternatively, it could be the expression levels of a constitutive genomic reporter in cells with fully active signaling and sensing modules.
o pg8, line 298-299; not clear why you are referring to Supplementary Figure 11 here. Actually, there seems to be an error in the numbering of Supplementary Figures; please correct this.
o pg8, ‘Cross-kingdom universality of the designed channels’; I am interested to see whether the same approach of promoter and strain design would work for the other communication channels that were presented (optional).
o pg8, line 320; refer to Fig4c,d where you refer to Supp Fig 16.
o pg9, line 336-340; show some diagram of the genetic circuit used to
implement the NOR gates. 

• Discussion
o pg9, line 354-356; please reword the first few sentences of the discussion. Strange English and potential to be misinterpreted or misunderstood.
o pg9, lines 362-368; missing referencing.
o Expand referencing in Discussion, possibly targeted to this field’s high impact
publications. 

• Methods
o Specify where verification by sequencing was performed. Specify how cell lines were verified for integration (optional).
o pg12, line 480; ‘white cell’ → ‘non-fluorescent cell’ replace
o pg12, line 485; include centrifugation speed and time throughout. 

• Figures
o Some figures have too small labels that are hard to read (e.g. Fig. 2, y-axes). o pg24, Fig 3; include quantitative measurements of data presented in the
heatmaps as a histogram showing actual values. The values are hard to read
off a color map. This can be presented in Supplementary materials. 

• Supplementary info
o Essential: Sequences of all developed genetic components and enzymes has to be available in Supplementary information in detail. A copiable sequence for all genetic tools that were used must be easily available.
o Much richer information on all utilized parts has to be included.
o All abbreviations have to be explained; e.g. Supp Fig 10 contains many abbreviations that are unexplained or ambiguous.
o Some figures have too small labels that are hard to read (e.g. Supp Fig 11). o Sequences should be copiable text.
o pg20: some explanation should be provided as to how the Sal system was
implemented into S. cerevisiae. If it was done analogously to the DAPG-PhlF system, this should be mentioned.




Reviewer #2:
Remarks to the Author:

The authors have described an approach to develop new communication pathways that can be
added to bacterial and eukaryotic cells that will enable molecular control over cells and cell
networks. In general, the article “hits the spot” in that there is a tremendous need for
advancements in this area and just a few innovative studies that have helped to define workable
solutions. The methodologies proposed here are rational; the importance is high and the example
methodology is noteworthy. My enthusiasm for this work is predicated on the need to solve
difficult issues concerning cell networks that exist in nature, particularly those were there is limited
analytical access and the need is great. I am thinking of the GI tract, for example. If we are to get
a handle on understanding the influence of the GI microbiome on human physiology, we will need
to engineer signaling among networks of cells and the community activities should be actuated in
non-invasive ways. Approaches such as those provided by the authors, while to me a logical
extension of earlier efforts, will be needed.

 The experiments appear to be well organized, executed, and with a few grammatical issues, well
 described. This reviewer is of the opinion that the overall methodology is robust. I have provided
 some thoughts below that will help with the presentation, justification, and novelty.

Primary thoughts:
The authors have based the work on requirements for (i) universality and (ii) orthogonality. The
need for universality seems valid in that the synthesis of signal molecules should be such that
exogenous precursors aren’t needed, instead typical biosynthetic functions should enable their
synthesis. In this way, the signals can be generated from a variety of organisms, in particular,
those that will be resident in the niche community of interest. The orthogonality argument has
been made by many and is reasonable here. In order to meet these requirements, constraints
were imposed, however, that limit the potential application and appeal. These should be either
discussed in more detail or otherwise validated.


1. One constraint is that the signal molecules freely diffuse across cell membranes. This
dramatically reduces the number of viable signal molecules and also limits the mode by which they
act in the host cells (i.e., binding to transcription factors). Was there are robust examination of
this limitation? Some additional discussion is warranted. For example, the types of pathways that
would be available for signal molecule generation is likely limited. Also, the gene circuits that can
be controlled using aTF’s will be limited. These constraints might have been buried, intentionally or
unintentionally, in the experimental results. For example, the authors validated the orthogonality
requirement by demonstrating minimal cross talk - they used the aTF’s that were an intrinsic part
of the study. There may have been other metabolic consequences that went undetected, but could
have ramifications relative to end use. Some discussion is warranted here relative to the number
of signal molecules, the types of pathways available for their synthesis and the limiting
consequences of using aTFs.

2. The authors have reduced the experimental range of the study by limiting the number of
enzymatic steps in the signal synthesis pathway. Specifically, they chose five enzymes as a
maximum and justified this by “reducing cellular resource taxing”. It would be important to show
how this was conceived. Currently, this seems to be an arbitrary decision. Some enzymes for
example, may represent a huge burden to the cells and could be in a two-step pathway, while a 7
step pathway might have minimal consequences based on flux through the pathway and the levels
of enzymes needed. Also, they have not provided any data associated with this constraint. If these
aren’t shown, then there is no need to suggest that it is fundamental to the approach. Rather, it
was a convenience.

3. Cross-kingdom universality. Why show this in HEK cells? Is there an intent to develop
methodologies for urinary tract infections? It would seem to be more important to show Cross
kingdom utility in a relevant cell line, such as an intestinal epithelial line. Provide some additional
rationale here.

4. The logic gate circuits. It would be helpful if the authors provide an example where these
complex circuits would be useful. The premise of the paper is on developing the molecules and
pathways. What computations are performed using these strains in logic gate circuits. This section
of the paper is limited to a paragraph and its utility could be strengthened.



Minor concerns (no particular order of importance):

L 49. Short- medium- long-range...perhaps better terms are autocrine, paracrine, endocrine...

L 54. This refers to “molecular communication” might cite this literature, especially as it relates to
 
L 66. Autonomous induction was first shown in 2010 (Tsao et al, Metabolic Engineering).

L 66. Autonomous population control last year (Stephens et al, Nature Comm.). Both of the above
exploit endogenous (AI-2) QS signaling.

L 82. AI-2 is an endogenous interkingdom signaling molecule (Zargar, mBio).

L 91. Missed recent pub on methods to develop orthogonal signals (McClune et al., Nature).

L 91. Presumes that the signaling molecules must pass through cell membranes and find their way
to aTFs. This is a considerable challenge, one met by the authors, and also a severe limitation on
the approach in that it presumes that the receiving cells have no metabolic activity for the signal
molecule (as noted above).

L 104. Some of them...state what it was...three?

L 126. Not sure I agree with the need to limit to 5 enzymes to reduce cellular taxing (as above).
The load of the signal generation itself could be distributed among cells. Moreover, if one enzyme
represents more of a burden than others, this would preclude this method from working. This is a
significant limitation, seemingly arbitrary. Perhaps the method should include an in silico method
for evaluating this criterion?

L 150. Two commas in a row. 

L 152. Several [of] the basal...

 L 170-180. This methodology is not novel. There is no discussion of the background level, just
 amplification (normalized). If there is appreciable background, this would erroneously be
 attributed to open communication.

L 187-198. This is a lot of work. Well done. It could alone be the topic of several papers. It is a
standard method, however.

L 190-220. Co-Cultures. How did you stimulate the synthesis of the signal molecule. It would seem
to be important to have a working system that opens a communication channel by providing a
stimulus. In the co-culture, the stimulus is important, then the time over which the sender cells
respond by making the signal molecule, the transport of the signal molecule to the receiver cells
(autocrine, paracrine, endocrine) and the subsequent transport into a response of the receiver
cells.

L251-L270. While this is a straightforward set of experiments and seems to have been expertly
executed, what is the point? Also, did you screen using sequence searches to make sure that there
aren’t any aTFs in the genomes of the supposed non-responding receivers?

L 289. Why did you transfer “some” and not all 10?

L 299. The transfer to HEK cells is very nice. It should be presented with more data and its own
 figure.

 L308-L350. Interesting work. This demonstrates the orthogonality of the signals and the sender
 and receiver cells. It is done on plates and depends on the diffusion of the signal molecules. I
wonder what the application of this might ever be?


Figures:
2. It is important to see the background levels. Suggest in supplemental providing the FACS data.
Specifically, one of the most important issues in studies like this has to do with the diffusion and
perception of the signal molecule. The fold induction, if performed using a fluorimeter, would give
you the total fluorescence. But, the number of cells might be affected as well as, or instead of, the
expression rate of the fluorophore in the receiving cell. Total fluorescence is the sum of both and
these are important. Please specify what was used here. If the number of cells that fluoresce is the
primary measure, then the transport of the signal molecule might have been a variable (that was
not accounted for).
3. I’m not sure why the YFP is normalized by RFP. We need amplification from zero not from a
different promoter.