Multiplexing cell‐cell communication

Abstract The engineering of advanced multicellular behaviors, such as the programmed growth of biofilms or tissues, requires cells to communicate multiple aspects of physiological information. Unfortunately, few cell‐cell communication systems have been developed for synthetic biology. Here, we engineer a genetically encoded channel selector device that enables a single communication system to transmit two separate intercellular conversations. Our design comprises multiplexer and demultiplexer sub‐circuits constructed from a total of 12 CRISPRi‐based transcriptional logic gates, an acyl homoserine lactone‐based communication module, and three inducible promoters that enable small molecule control over the conversations. Experimentally parameterized mathematical models of the sub‐components predict the steady state and dynamical performance of the full system. Multiplexed cell‐cell communication has applications in synthetic development, metabolic engineering, and other areas requiring the coordination of multiple pathways among a community of cells.

Thank you again for submitting your work to Molecular Systems Biology. We have now heard back from the reviewer who was asked to evaluat e your st udy. As we previously discussed, the reviewer was given access to the manuscript and your responses to the reviewers' comment s from the other journal. They were asked to evaluate whether the reviewers' concerns have been adequat ely addressed, and to assess the suit abilit y of the st udy for publicat ion keeping in mind the edit orial crit eria of Molecular Syst ems Biology. As you will see below, the reviewer thinks that you have done an excellent job in addressing the previous concerns and is support ive of publicat ion, pending some very minor modificat ions. Sext on and Tabor engineered a channel select or synt het ic gene circuit that swit ches bet ween two int ercellular conversat ions. The aut hors exhaust ively test ed it s funct ionalit y in E. coli and demonst rat ed that the circuit is act uat ed as desired. The circuit is comprised of 12 CRISPRi-based transcript ional logic gat es, 3 inducible promot ers, and acryl homoserine lact one (AHL)-mediat ed quorum sensing syst em.
Borrowing it s concept from elect rical engineering, the present ed genet ically encoded channel select or links a mult iplexer (MUX) and demult iplexer (DEMUX) circuit . MUX and DEMUX are const ruct ed using several ort hogonal NOT and NOR logic gat es. They are implement ed in separat e bact erial st rains, charact erized individually, and co-cult ured to validat e their full funct ionalit y. The aut hors also demonst rat e that their circuit is capable of swit ching bet ween the two conversat ion dynamically.
All of the circuit behavior aligns wit h the aut hors' mat hemat ical predict ions, which furt her support the validit y of the circuit funct ion. The aut hors have done a very thorough job in responding to the comment s/suggest ions by the init ial reviewers. Overall, I'm impressed wit h the work and support it s publicat ion.
Minor comment s 1. The complexity, integrity, and performance of the circuit is fascinating. The authors did a stunning amount of work thoroughly characterizing and validating various components.
2. Because of the nature of the work, it can be confusing to follow through several negations in signal concentrations. It will help clarify if the authors could clearly state that inducer concentrations are not the inputs (IN1 or IN2) but instead the NOT(inducer) is the input to the channel selector.

2nd Jun 2020 1st Authors' Response to Reviewers
We are happy to hear that the reviewer is positive about our manuscript and the edits we have made in response to the previous reviewers, and supports publication in Molecular Systems Biology.
In response to the new reviewer's second comment, we have changed the beginning of the "Linking the MUX to small-molecule inducers and the cell-cell communication system" section from: "To this end, we first constructed SENSOR-MUX-AHL, wherein the aTc sensor controls IN1, an isopropyl β-D-1-thiogalactopyranoside (IPTG) sensor controls IN2, and a 2,4diacetylphloroglucinol (DAPG) sensor controls SELECT, each via an additional sgRNA ( Thank you for performing the requested text changes. We are now satisfied with the modifications made and I am pleased to inform you that your paper has been accepted for publication. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.

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Corresponding Author Name: Jeffrey J. Tabor

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