Genetic Circuitry Research Articles

A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

Electrolocation? The evidence for redox-mediated taxis in Shewanella oneidensis.

Shewanella oneidensis is a dissimilatory metal reducing bacterium and model for extracellular electron transfer (EET), a respiratory mechanism in which electrons are transferred out of the cell. In the last 10years, migration to insoluble electron acceptors for EET has been shown to be nonrandom and tactic, seemingly in the absence of molecular or energy gradients that typically allow for taxis. As the ability to sense, locate, and respire electrodes has applications in bioelectrochemical technology, a better understanding of taxis in S. oneidensis is needed. While the EET conduits of S. oneidensis have been studied extensively, its taxis pathways and their interplay with EET are not yet understood, making investigation into taxis phenomena nontrivial. Since S. oneidensis is a member of an EET-encoding clade, the genetic circuitry of taxis to insoluble acceptors may be conserved. We performed a bioinformatic analysis of Shewanella genomes to identify S. oneidensis chemotaxis orthologs conserved in the genus. In addition to the previously reported core chemotaxis gene cluster, we identify several other conserved proteins in the taxis signaling pathway. We present the current evidence for the two proposed models of EET taxis, "electrokinesis" and flavin-mediated taxis, and highlight key areas in need of further investigation.

One stem cell program to rule them all?

Many species of animals have stem cells that they maintain throughout their lives, which suggests that stem cells are an ancestral feature of all animals. From this, we take the viewpoint that cells with the biological properties of 'stemness'-self-renewal and multipotency-may share ancestral genetic circuitry. However, in practice is it very difficult to identify and compare stemness gene signatures across diverse animals and large evolutionary distances? First, it is critical to experimentally demonstrate self-renewal and potency. Second, genomic methods must be used to determine specific gene expression in stem cell types compared with non-stem cell types to determine stem cell gene enrichment. Third, gene homology must be mapped between diverse animals across large evolutionary distances. Finally, conserved genes that fulfill these criteria must be tested for role in stem cell function. It is our viewpoint that by comparing stem cell-specific gene signatures across evolution, ancestral programs of stemness can be uncovered, and ultimately, the dysregulation of stemness programs drives the state of cancer stem cells.

The Cis-Regulatory Code for Kelch-like 21/30 Specific Expression in Ciona robusta Sensory Organs.

The tunicate Ciona robusta is an emerging model system to study the evolution of the nervous system. Due to their small embryos and compact genomes, tunicates, like Ciona robusta, have great potential to comprehend genetic circuitry underlying cell specific gene repertoire, among different neuronal cells. Their simple larvae possess a sensory vesicle comprising two pigmented sensory organs, the ocellus and the otolith. We focused here on Klhl21/30, a gene belonging to Kelch family, that, in Ciona robusta, starts to be expressed in pigmented cell precursors, becoming specifically maintained in the otolith precursor during embryogenesis. Evolutionary analyses demonstrated the conservation of Klhl21/30 in all the chordates. Cis-regulatory analyses and CRISPR/Cas9 mutagenesis of potential upstream factors, revealed that Klhl21/30 expression is controlled by the combined action of three transcription factors, Mitf, Dmrt, and Msx, which are downstream of FGF signaling. The central role of Mitf is consistent with its function as a fundamental regulator of vertebrate pigment cell development. Moreover, our results unraveled a new function for Dmrt and Msx as transcriptional co-activators in the context of the Ciona otolith.

General Analyses of Gene Expression Dependencies on Genetic Burden

Over the last decade, the combining of newly developed molecular tools for DNA editing with engineering principles has allowed the creation of complex cellular devices, usually based on complex genetic circuits, for many different purposes. However, when the technological evolution of genetic circuitry is compared with previous technologies such as electronic circuitry, clear limitations regarding the technological scalability of genetic circuitry are observed due to the lack of predictability. To overcome this problem, it is necessary to create new theoretical frameworks for designing genetic circuits in a feasible and reliable manner, taking into account those limitations. Among a number of such limitations, the so-called genetic burden is one of the main constraints. Surprisingly, despite its relevance, little attention has been paid to genetic burden, and it is often not considered when designing genetic circuits. In this study, a new general mathematical formalism is presented, describing the effects of genetic burden on gene expression. The mathematical analysis shows that alterations in gene expression due to genetic burden can be qualitatively described independently of the specific genetic features of the system under consideration. The mathematical model was experimentally tested in different genetic circuits. The experimental evidence coincides with the expected behaviors described by the model in complex scenarios. For instance, observed modulations in the expression levels of constitutive genes in response to changes in the levels of external inducers of gene expression that do not directly modulate them, or the emergence of limitations in gene overexpression, can be understood in terms of genetic burden. The present mathematical formalism provides a useful general framework for gene circuit design that will help to advance synthetic biological systems.

Conservation as well as divergence in Mcidas function underlies the differentiation of multiciliated cells in vertebrates

Multiciliated cells (MCCs) differentiate hundreds of motile cilia that beat to drive fluid movement over various kinds of epithelia. In Xenopus, mice and human, the coiled-coil containing protein Mcidas (Mci) has been shown to be a key transcriptional regulator of MCC differentiation. We have examined Mci function in the zebrafish, another model organism that is widely used to study ciliary biology. We show that zebrafish mci is expressed specifically in the developing MCCs of the kidney tubules, but surprisingly, not in those of the nasal placodes. Mci proteins lack a DNA binding domain and associate with the cell-cycle transcription factors E2f4/5 for regulating MCC-specific gene expression. We found that while the zebrafish Mci protein can complex with the E2f family members, its sequence as well as the requirement and sufficiency for MCC differentiation has diverged significantly from Mci homologues of the tetrapods. We also provide evidence that compared to Gmnc, another related coiled-coil protein that has recently been shown to regulate MCC development upstream of Mci, the Mci protein originated later within the vertebrate lineage. Based on these data, we argue that in contrast to Gmnc, which has a vital role in the genetic circuitry that drives MCC formation, the requirement of Mci, at least in the zebrafish, is not obligatory.

Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target.

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a devastating neurodegenerative disorder lacking effective treatments. ALS pathology is linked to mutations in >20 different genes indicating a complex underlying genetic architecture that is effectively unknown. Here, in an attempt to identify genes and pathways for potential therapeutic intervention and explore the genetic circuitry underlying Drosophila models of ALS, we carry out two independent genome-wide screens for modifiers of degenerative phenotypes associated with the expression of transgenic constructs carrying familial ALS-causing alleles of FUS (hFUSR521C) and TDP-43 (hTDP-43M337V). We uncover a complex array of genes affecting either or both of the two strains, and investigate their activities in additional ALS models. Our studies indicate the pathway that governs phospholipase D activity as a major modifier of ALS-related phenotypes, a notion supported by data we generated in mice and others collected in humans.

Stochastic ordering of complexoform protein assembly by genetic circuits

Top-down proteomics has enabled the elucidation of heterogeneous protein complexes with different cofactors, post-translational modifications, and protein membership. This heterogeneity is believed to play a previously unknown role in cellular processes. The different molecular forms of a protein complex have come to be called "complex isoform" or "complexoform". Despite the elucidation of the complexoform, it remains unclear how and whether cellular circuits control the distribution of a complexoform. To help address this issue, we first simulate a generic three-protein complexoform to reveal the control of its distribution by the timing of gene transcription, mRNA translation, and protein transport. Overall, we ran 265 computational experiments: each averaged over 1,000 stochastic simulations. Based on the experiments, we show that genes arranged in a single operon, a cascade, or as two operons all give rise to the different protein composition of complexoform because of timing differences in protein-synthesis order. We also show that changes in the kinetics of expression, protein transport, or protein binding dramatically alter the distribution of the complexoform. Furthermore, both stochastic and transient kinetics control the assembly of the complexoform when the expression and assembly occur concurrently. We test our model against the biological cellulosome system. With biologically relevant rates, we find that the genetic circuitry controls the average final complexoform assembly and the variation in the assembly structure. Our results highlight the importance of both the genetic circuit architecture and kinetics in determining the distribution of a complexoform. Our work has a broad impact on our understanding of non-equilibrium processes in both living and synthetic biological systems.

The Genetic and Neural Circuitry Predictors of Benefit From Manualized or Open-Ended Psychotherapy.

New technologies incorporating genetics and neuroimaging into psychiatric care offer the possibility of illuminating associations among genetic alleles, neural functioning, and patients' response to various psychotherapeutic modalities. In this review, the authors survey the literature on the emerging field of genetic predictors of psychotherapy response, particularly in relation to the 5-HTTLPR polymorphism and individual response to manualized psychotherapy. The extant literature was reviewed, with PubMed serving as the primary database. Several polymorphisms have been linked with response or resistance to treatment. Given the number of studies assessing the relevance of the 5-HTTLPR polymorphism to treatment response, this review focuses on this genetic variation. Because individual genetic endowments may predict nonresponse to manualized treatment modalities, it may become possible to identify individuals who would benefit from insight-oriented, open-ended psychotherapy tailored to their individual distress tolerance levels, rather than from shorter manualized treatment.

Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum.

Chromobacterium violaceum has been of interest recently due to its cyanogenic ability and its potential role in environmental sustainability via the biorecovery of gold from electronic waste. However, as with many nonmodel bacteria, there are limited genetic tools to implement the use of this Gram-negative chassis in synthetic biology. We propose a system that involves assaying spontaneous antibiotic resistances and using broad host range vectors to develop episomal vectors for nonmodel Gram-negative bacteria. These developed vectors can subsequently be used to characterize inducible promoters for gene expressions and implementing CRISPRi to inhibit endogenous gene expression for further studies. Here, we developed the first episomal genetic toolkit for C. violaceum consisting of two origins of replication, three antibiotic resistance genes, and four inducible promoter systems. We examined the occurrences of spontaneous resistances of the bacterium to the chosen selection markers to prevent incidences of false positives. We also tested broad host range vectors from four different incompatibility groups and characterized four inducible promoter systems, which potentially can be applied in other Gram-negative nonmodel bacteria. CRISPRi was also implemented to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building in this chassis and other nonmodel bacteria for synthetic biology and biotechnological applications.

Deconvoluting Wavelengths Leading to Fluorescent Light Induced Inflammation and Cellular Stress in Zebrafish (Danio rerio)

Fluorescent light (FL) has been shown to induce a cellular immune and inflammatory response that is conserved over 450 MY of evolutionary divergence and among vertebrates having drastically different lifestyles such as Mus musculus, Danio rerio, Oryzias latipes and Xiphophorus maculatus. This surprising finding of an inflammation and immune response to FL not only holds for direct light receiving organs (skin) but is also observed within internal organs (brain and liver). Light responsive genetic circuitry initiated by the IL1B regulator induces a highly conserved acute phase response in each organ assessed for all of biological models surveyed to date; however, the specific light wavelengths triggering this response have yet to be determined so investigation of mechanisms and/or light specific molecule(s) leading to this response are difficult to assess. To understand how specific light wavelengths are received in both external and internal organs, zebrafish were exposed to specific 50 nm light wavebands spanning the visible spectrum from 300–600 nm and the genetic responses to each waveband exposure were assessed. Surprisingly, the induced cellular stress response previously observed following FL exposure is not triggered by the lower “damaging” wavelengths of light (UVB and UVA from 300–400 nm) but instead is maximally induced by higher wavelengths ranging from 450–500 nm in skin to 500–600 nm in both brain and liver).

A novel genetic circuitry governing hypoxic metabolic flexibility, commensalism and virulence in the fungal pathogen Candida albicans.

Inside the human host, the pathogenic yeast Candida albicans colonizes predominantly oxygen-poor niches such as the gastrointestinal and vaginal tracts, but also oxygen-rich environments such as cutaneous epithelial cells and oral mucosa. This suppleness requires an effective mechanism to reversibly reprogram the primary metabolism in response to oxygen variation. Here, we have uncovered that Snf5, a subunit of SWI/SNF chromatin remodeling complex, is a major transcriptional regulator that links oxygen status to the metabolic capacity of C. albicans. Snf5 and other subunits of SWI/SNF complex were required to activate genes of carbon utilization and other carbohydrates related process specifically under hypoxia. snf5 mutant exhibited an altered metabolome reflecting that SWI/SNF plays an essential role in maintaining metabolic homeostasis and carbon flux in C. albicans under hypoxia. Snf5 was necessary to activate the transcriptional program linked to both commensal and invasive growth. Accordingly, snf5 was unable to maintain its growth in the stomach, the cecum and the colon of mice. snf5 was also avirulent as it was unable to invade Galleria larvae or to cause damage to human enterocytes and murine macrophages. Among candidates of signaling pathways in which Snf5 might operate, phenotypic analysis revealed that mutants of Ras1-cAMP-PKA pathway, as well as mutants of Yak1 and Yck2 kinases exhibited a similar carbon flexibility phenotype as did snf5 under hypoxia. Genetic interaction analysis indicated that the adenylate cyclase Cyr1, a key component of the Ras1-cAMP pathway interacted genetically with Snf5. Our study yielded new insight into the oxygen-sensitive regulatory circuit that control metabolic flexibility, stress, commensalism and virulence in C. albicans.

Dosage analysis of the 7q11.23 Williams region identifies BAZ1B as a major human gene patterning the modern human face and underlying self-domestication.

We undertook a functional dissection of chromatin remodeler BAZ1B in neural crest (NC) stem cells (NCSCs) from a uniquely informative cohort of typical and atypical patients harboring 7q11.23 copy number variants. Our results reveal a key contribution of BAZ1B to NCSC in vitro induction and migration, coupled with a crucial involvement in NC-specific transcriptional circuits and distal regulation. By intersecting our experimental data with new paleogenetic analyses comparing modern and archaic humans, we found a modern-specific enrichment for regulatory changes both in BAZ1B and its experimentally defined downstream targets, thereby providing the first empirical validation of the human self-domestication hypothesis and positioning BAZ1B as a master regulator of the modern human face. In so doing, we provide experimental evidence that the craniofacial and cognitive/behavioral phenotypes caused by alterations of the Williams-Beuren syndrome critical region can serve as a powerful entry point into the evolution of the modern human face and prosociality.

Selective breeding modifies mef2ca mutant incomplete penetrance by tuning the opposing Notch pathway.

Deleterious genetic mutations allow developmental biologists to understand how genes control development. However, not all loss of function genetic mutants develop phenotypic changes. Many deleterious mutations only produce a phenotype in a subset of mutant individuals, a phenomenon known as incomplete penetrance. Incomplete penetrance can confound analyses of gene function and our understanding of this widespread phenomenon remains inadequate. To better understand what controls penetrance, we capitalized on the zebrafish mef2ca mutant which produces craniofacial phenotypes with variable penetrance. Starting with a characterized mef2ca loss of function mutant allele, we used classical selective breeding methods to generate zebrafish strains in which mutant-associated phenotypes consistently appear with low or high penetrance. Strikingly, our selective breeding for low penetrance converted the mef2ca mutant allele behavior from homozygous lethal to homozygous viable. Meanwhile, selective breeding for high penetrance converted the mef2ca mutant allele from fully recessive to partially dominant. Comparing the selectively-bred low- and high-penetrance strains revealed that the strains initially respond similarly to the mutation, but then gene expression differences between strains emerge during development. Thus, altered temporal genetic circuitry can manifest through selective pressure to modify mutant penetrance. Specifically, we demonstrate differences in Notch signaling between strains, and further show that experimental manipulation of the Notch pathway phenocopies penetrance changes occurring through selective breeding. This study provides evidence that penetrance is inherited as a liability-threshold trait. Our finding that vertebrate animals can overcome a deleterious mutation by tuning genetic circuitry complements other reported mechanisms of overcoming deleterious mutations such as transcriptional adaptation of compensatory genes, alternative mRNA splicing, and maternal deposition of wild-type transcripts, which are not observed in our system. The selective breeding approach and the resultant genetic circuitry change we uncovered advances and expands our current understanding of genetic and developmental resilience.

A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition.

Integrating single-cell trajectory analysis with pooled genetic screening could reveal the genetic architecture that guides cellular decisions in development and disease. We applied this paradigm to probe the genetic circuitry that controls epithelial-to-mesenchymal transition (EMT). We profiled epithelial cells undergoing a spontaneous, spatially determined EMT in the presence or absence of TGF-β via single-cell RNA-seq. Pseudospatial trajectory analysis identified continuous waves of gene regulation, as opposed to discrete “partial” stages of EMT. KRAS was connected to exit from the epithelial state and acquisition of a fully mesenchymal phenotype. A pooled single-cell CRISPR-Cas9 screen identified EMT-associated receptors and transcription factors, including regulators of KRAS, whose loss impeded progress along EMT. Inhibiting the KRAS effector MEK, and its upstream activators EGFR and MET, demonstrates that interruption of key signaling events reveals regulatory “checkpoints” in the EMT continuum that mimic discrete stages and reconciles opposing views of the program that controls EMT.

105-OR: Lineage Determination of Endocrine Progenitors in Murine and Human Pancreatic Development

Generation of functional beta cells for regenerative medicine would benefit from an increased understanding of pancreatic endocrine cell differentiation. During pancreatic development, expression of Ngn3 defines the progenitor cells that will differentiate into one of five endocrine lineages, including the beta cell. Here, we identify and further characterize a distinct endocrine progenitor population defined by differential expression of a transcription factor named Fev in murine and human endocrine cell development. Using single-cell RNA-sequencing and genetic lineage tracing, we build a transcriptomic profile of murine Fev+ progenitors that is distinct from that of Ngn3+ progenitors and reconstruct lineage relationships among Ngn3+, Fev+, and differentiated, hormone+ cells. We further identify candidate transcriptional regulators along the differentiation trajectory of this Fev+ progenitor population toward the alpha or beta cell lineages. Translating our findings in murine pancreatic development to that of human, we identify an analogous FEV+ population and putative gene regulators of endocrine cell fate in human fetal pancreas. In a stepwise differentiation platform designed to generate beta cells from human embryonic stem cells (hESCs), we also uncover robust FEV expression in endocrine progenitors prior to their differentiation into insulin-producing beta cells. Single-cell RNA-sequencing and lineage reconstruction of hESC-derived cells at the differentiated beta cell stage reveal FEV+ cells that may represent endocrine progenitors that have failed to acquire a beta cell identity; this failure of FEV+ endocrine cell differentiation may facilitate in uncovering the required molecular signals and genetic circuitry that push endocrine progenitors toward a beta cell fate. This work establishes a new model of pancreatic endocrine cell differentiation and identifies a Fev+ endocrine progenitor as a new cell stage in endocrine lineage determination. Disclosure D.M. Wong: None. L. Byrnes: None. J.O. Bouza: None. G. Peixoto: None. R. McMullen: None. J.B. Sneddon: Advisory Panel; Spouse/Partner; Akouos. Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Consultant; Self; Semma Therapeutics, Inc. Funding Nora Eccles Treadwell Foundation; University of California, San Francisco

Pheromones and Nutritional Signals Regulate the Developmental Reliance on let-7 Family MicroRNAs in C. elegans

Adverse environmental conditions can affect rates ofanimal developmental progression and lead to temporary developmental quiescence (diapause), exemplified by the dauer larva stage of the nematode Caenorhabditis elegans (C.elegans). Remarkably, patterns of cell division and temporal cell-fate progression in C.elegans larvae are not affected by changes in developmental trajectory. However, the underlying physiological and gene regulatory mechanisms that ensure robust developmental patterning despite substantial plasticity in developmental progression are largely unknown. Here, we report thatdiapause-inducing pheromones correct heterochronic developmental cell lineage defects caused by insufficient expression of let-7 family microRNAs in C.elegans. Moreover, two conserved endocrine signaling pathways, DAF-7/TGF-β and DAF-2/Insulin, that confer on the larva diapause and non-diapause alternative developmental trajectories interact with the nuclear hormone receptor, DAF-12, to initiate and regulate a rewiring of the genetic circuitry controlling temporal cell fates. This rewiring includes engagement of certain heterochronic genes, lin-46, lin-4, and nhl-2, that are previously associated with an altered genetic program in post-diapause animals, in combination with a novel ligand-independent DAF-12 activity, to downregulate the critical let-7 family target Hunchback-like-1 (HBL-1). Our results show how pheromone or endocrine signaling pathways can coordinately regulate both developmental progression and cell-fate transitions in C.elegans larvae under stress so that the developmental schedule of cell fates remains unaffected by changes in developmental trajectory.

An Nfil3-Zeb2-Id2 pathway imposes Irf8 enhancer switching during cDC1 development

Abstract Classical type 1 dendritic cells (cDC1) are required for anti-viral and anti-tumor immunity, motivating a need to understand their development. We recently discovered that the development of the cDC1 progenitor requires an E protein-dependent +41 kb Irf8 enhancer, but its maturation instead requires a BATF3-dependent +32 kb Irf8 enhancer. To identify the genetic circuitry underlying this switch, we performed single-cell RNA-sequencing of the multipotent common dendritic cell progenitor (CDP). We found a cluster of cells within the CDP that already expressed transcription factors influencing cDC1 development, including Nfil3, Id2, and Zeb2. Genetic epistasis between these factors revealed a hierarchy of action in which Nfil3 expression is required for a transition from Zeb2hi and Id2lo CDPs into Zeb2lo and Id2hi CDPs, which represent the earliest committed cDC1 progenitors. This genetic circuit blocks E protein activity to exclude pDC potential within this progenitor and also explains the switch in Irf8 enhancer usage during cDC1 development.

Transcriptomics analysis defines global cellular response of Agrobacterium tumefaciens 5A to arsenite exposure regulated through the histidine kinases PhoR and AioS.

In environments where arsenic and microbes coexist, microbes are the principal drivers of arsenic speciation, which directly affects bioavailability, toxicity and bioaccumulation. Speciation reactions influence arsenic behaviour in environmental systems, directly affecting human and agricultural exposures. Arsenite oxidation decreases arsenic toxicity and mobility in the environment, and therefore understanding its regulation and overall influence on cellular metabolism is of significant interest. The arsenite oxidase (AioBA) is regulated by a three-component signal transduction system AioXSR, which is in turn regulated by the phosphate stress response, with PhoR acting as the master regulator. Using RNA-sequencing, we characterized the global effects of arsenite on gene expression in Agrobacterium tumefaciens 5A. To further elucidate regulatory controls, mutant strains for histidine kinases PhoR and AioS were employed, and illustrate that in addition to arsenic metabolism, a host of other functional responses are regulated in parallel. Impacted functions include arsenic and phosphate metabolism, carbohydrate metabolism, solute transport systems and iron metabolism, in addition to others. These findings contribute significantly to the current understanding of the metabolic impact and genetic circuitry involved during arsenite exposure in bacteria. This informs how arsenic contamination will impact microbial activities involving several biogeochemical cycles in nature.

A Self-Actuated Cellular Protein Delivery Machine.

Engineered bacterial cells have great promise to solve global problems, yet they are hampered by a lack of convenient strategy for controlled protein release. A well-controlled protein translocation through cellular membranes is essential for cell-based protein delivery. Here we have developed a controlled protein release system by programming a bacterial autotransporter system named Ag43. Ag43 protein is engineered by adding a protease digestion site between its translocation and cargo domains. Once it is displayed on the cell surface, we managed to release the cargo proteins in defined conditions by processing environmental signals. The protein release in terms of time and quantity can be controlled through changing the inducer conditions. We thought that the release system can be adopted for complex genetic circuitries due to its simplicity. We implemented the protein release system to develop a cellular device that is able to release proteins in a sequence response to ordered chemical signals. We envision that development of genetically controlled protein release systems will improve the applications of synthetic organisms in cell based therapies, especially for cases with a need for controlled protein release using the cues from the biological environment.