Technical Report on "Limitations and trade-offs in gene expression due to competition for shared cellular resources"

This is a technical report accompanying the paper entitled “Limitations and trade-offs in gene expression due to competition for shared cellular resources”.This work was supported by AFOSR grant FA9550-12-1-0129 and NIGMS grant P50 GM098792

Limitations and trade-offs in gene expression due to competition for shared cellular resources

Gene circuits share transcriptional and translational resources in the cell. The fact that these common resources are available only in limited amounts leads to unexpected couplings in protein expressions. As a result, our predictive ability of describing the behavior of gene circuits is limited. In this paper, we consider the simultaneous expression of proteins and describe the coupling among protein concentrations due to competition for RNA polymerase and ribosomes. In particular, we identify the limitations and trade-offs in gene expression by characterizing the attainable combinations of protein concentrations. We further present two application examples of our results: we show that even in the absence of regulatory linkages, genes can seemingly behave as repressors, and surprisingly, as activators to each other, purely due to the limited availability of shared cellular resources.United States. Air Force Office of Scientific Research (Grant FA9550-12-1-0129)National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792

Characterization and mitigation of gene expression burden in mammalian cells

Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells

Contextualizing context for synthetic biology--identifying causes of failure of synthetic biological systems.

Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo. In this review, we set out to identify and classify the sources of the unexpected divergences between design and actual function of synthetic systems and analyze possible methodologies aimed at controlling, if not preventing, unwanted contextual issues

RBS and Promoter Strengths Determine the Cell-Growth-Dependent Protein Mass Fractions and Their Optimal Synthesis Rates

[EN] Models of gene expression considering host-circuit interactions are relevant for understanding both the strategies and associated trade-offs that cell endogenous genes have evolved and for the efficient design of heterologous protein expression systems and synthetic genetic circuits. Here, we consider a small-size model of gene expression dynamics in bacterial cells accounting for host-circuit interactions due to limited cellular resources. We define the cellular resources recruitment strength as a key functional coefficient that explains the distribution of resources among the host and the genes of interest and the relationship between the usage of resources and cell growth. This functional coefficient explicitly takes into account lab-accessible gene expression characteristics, such as promoter and ribosome binding site (RBS) strengths, capturing their interplay with the growth-dependent flux of available free cell resources. Despite its simplicity, the model captures the differential role of promoter and RBS strengths in the distribution of protein mass fractions as a function of growth rate and the optimal protein synthesis rate with remarkable fit to the experimental data from the literature for Escherichia coli. This allows us to explain why endogenous genes have evolved different strategies in the expression space and also makes the model suitable for model-based design of exogenous synthetic gene expression systems with desired characteristics.This work was partially supported by grants MINECO/AEI, EU DPI2017-82896-C2-1-R, and MCIN/AEI/10.13039/501100011033 grant number PID2020-117271RB-C21. F.N.S.-N. is grateful to grant PAID-01-2017 (Universitat Politecnica de Valencia). The authors are very grateful to the anonymous reviewers for their comprehensive and in-depth reviews.Santos-Navarro, FN.; Vignoni, A.; Boada-Acosta, YF.; Picó, J. (2021). RBS and Promoter Strengths Determine the Cell-Growth-Dependent Protein Mass Fractions and Their Optimal Synthesis Rates. ACS Synthetic Biology. 10(12):3290-3303. https://doi.org/10.1021/acssynbio.1c0013132903303101

Prediction of Cellular Burden with Host--Circuit Models

Heterologous gene expression draws resources from host cells. These resources include vital components to sustain growth and replication, and the resulting cellular burden is a widely recognised bottleneck in the design of robust circuits. In this tutorial we discuss the use of computational models that integrate gene circuits and the physiology of host cells. Through various use cases, we illustrate the power of host-circuit models to predict the impact of design parameters on both burden and circuit functionality. Our approach relies on a new generation of computational models for microbial growth that can flexibly accommodate resource bottlenecks encountered in gene circuit design. Adoption of this modelling paradigm can facilitate fast and robust design cycles in synthetic biology

The influence of biological rhythms on host–parasite interactions

Biological rhythms, from circadian control of cellular processes to annual cycles in life history, are a main structural element of biology. Biological rhythms are considered adaptive because they enable organisms to partition activities to cope with, and take advantage of, predictable fluctuations in environmental conditions. A flourishing area of immunology is uncovering rhythms in the immune system of animals, including humans. Given the temporal structure of immunity, and rhythms in parasite activity and disease incidence, we propose that the intersection of chronobiology, disease ecology, and evolutionary biology holds the key to understanding host–parasite interactions. Here, we review host–parasite interactions while explicitly considering biological rhythms, and propose that rhythms: influence within-host infection dynamics and transmission between hosts, might account for diel and annual periodicity in host–parasite systems, and can lead to a host–parasite arms race in the temporal domain

Analysis of transcriptional feedback strategy for reducing interaction in gene expression processes

Advances in genetic manipulation have allowed the overexpression of proteins and insertion of circuits in cells. However, the expected behaviour can be altered by the internal competition for the limited amount of cellular resources. In this work we analyse a feedback strategy based on transcription inhibition that aims to reduce the interaction in a two-protein expression system. The results allow interpreting the effects of negative feedback on the steady-state protein levels and how the realizable protein region is affected by the feedback loop.Fil: Nuñez, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones en Electrónica, Control y Procesamiento de Señales. Universidad Nacional de La Plata. Instituto de Investigaciones en Electrónica, Control y Procesamiento de Señales; ArgentinaFil: Garelli, Fabricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones en Electrónica, Control y Procesamiento de Señales. Universidad Nacional de La Plata. Instituto de Investigaciones en Electrónica, Control y Procesamiento de Señales; ArgentinaFil: Picó, Jesús. Universidad Politécnica de Valencia. Departamento de Ingeniería de Sistemas y Automática; EspañaFil: de Battista, Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones en Electrónica, Control y Procesamiento de Señales. Universidad Nacional de La Plata. Instituto de Investigaciones en Electrónica, Control y Procesamiento de Señales; Argentin

Energetic Cost and Physiological Trade-offs

Understanding how organisms allocate limited resources across physiological systems is a major challenge in biology. This study revealed that high energetic demand of electric signals of male electric fish (Brachyhypopomus gauderio) is matched by a metabolic trade-off with other cellular functions. We used thyroxine (T4) to modulate the fish’s signal metabolism, partitioned the energy budget pharmacologically, and measured energy consumption using oxygen respirometry. In males, total energy consumption was unchanged pre- and post-T4 treatment, while signal metabolism rose and the standard metabolic rate fell in an even trade-off. Total metabolism in females did the opposite. Under T4, the non-signal resting metabolism rose while the signal metabolismfell. These results reveal sex differences in metabolic trade-offs between signaling and cellular metabolism in electric fish and suggest that thyroid hormones regulate the allocation of energy between electric signals and somatic maintenance in favor of reproduction. To determine whether electric fish trade-off reproduction against innate immunity, as is common in vertebrates, we assessed changes in the bactericidal activity of plasma in B. gauderio challenged with bacterial lipopolysaccharide (LPS), before and after T4 treatment. Females did not modulate innate immunity with any of the treatments, while males elevated bactericidal activity of plasma by about a third following LPS injections, T4 implants, or both together, relative to sham treatment. This outcome was unexpected given that T4 increases the energy consumed by the male’s reproductive electric signals while lowering the rest of his metabolism. T4 also increased expression of Na+K+ATPase pump mRNA in the electrogenic cells of males but not females, consistent with previous findings that T4 differentially regulates signal metabolism in the two sexes. This sex difference in gene regulation suggests Na+K+ATPase underlies sexual dimorphism in electric signal energetics. The results provide further evidence that thyroid hormones play an essential role in the differential allocation of energy among metabolic functions. This body of work is the first to quantify an energetic trade-off between reproductive behavior and other metabolic functions. and implicates ion pumps, but not innate immunity, as molecular mechanisms underlying sex differences found in these energetic trade-offs