Feedforward ribosome control mitigates gene activation burden

Barajas, C. A. Chavez; Gibson, Jesse; Sandoval, Luis; Vecchio, Domitilla Del

doi:10.1101/2021.02.11.430724

Cited by 5 publications

(6 citation statements)

References 114 publications

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“…In turn, the operation of genetic devices can change the cell's growth rate (Ceroni et al, 2015;Jones et al, 2020). One way to approach the problem of genetic devices affecting cell growth rates has been to use feedforward and/or feedback controllers to limit the burden of genetic devices on the bacterial (Barajas et al, 2021;Ceroni et al, 2018) or mammalian cell (Lillacci et al, 2018) (Figures 9Aii and 9Cii), thus better optimizing the resource usage of a genetic device given the constraints of the cellular environment. Note that the (Huang et al, 2018).…”

Section: Applications Of Controllers To Insulate Genetic Device Function From Contextmentioning

confidence: 99%

“…These designs have also enabled adaptation to DNA copy number (Bleris et al, 2011;Jones et al, 2020;Lillacci et al, 2018), as well as resource competition (Frei et al, 2020a;Jones et al, 2020), transcriptional inducers (Strovas et al, 2014), and changes in protease production (Gao et al, 2018). (Cv) A recent study demonstrated feedforward control of ribosome levels to offset loading of the ribosome by the output protein by co-expressing SpoTH, the hydrolysis domain of SpoT, a positive regulator of ribosome activity in bacteria (Barajas et al, 2021). This was shown to reduce the effects of resource loading both on other genes and on cell growth rates.…”

Section: Applications Of Controllers To Insulate Genetic Device Function From Contextmentioning

confidence: 99%

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Context-aware synthetic biology by controller design: Engineering the mammalian cell

2021

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Section: Applications Of Controllers To Insulate Genetic Device Function From Contextmentioning

confidence: 99%

Section: Applications Of Controllers To Insulate Genetic Device Function From Contextmentioning

confidence: 99%

Context-aware synthetic biology by controller design: Engineering the mammalian cell

2021

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“…Barajas et al utilized a similar global architecture of circuit-wide activation (rather than repression) by coexpressing the hydrolysis domain of SpoTH to upregulate free ribosome production with the gene of interest, resulting in sufficient resources to overcome the increased resource load of the gene of interest. 27 In the LC archetype, on the other hand, each controller node represses only the gene it is regulating. Jones et al also demonstrated the efficacy of this controller type by coexpressing an endoribonuclease along with the gene-of-interest, which targets the mRNA of gene-of-interest, thereby mitigating the resource consumption of the gene-of-interest via a local feedforward control mechanism.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Incoherent feedforward loops (iFFLs) couple both activation and repression arms and have been implicated to function in adaptation behavior and pulse generation. − They have also been demonstrated to function as resource regulators − due to the repressive links inherent to the iFFL architecture, much like the negative feedback motif. Here, we extend our analysis of the aforementioned three controller architectures to iFFLs.…”

Section: Introductionmentioning

confidence: 99%

Negatively Competitive Incoherent Feedforward Loops Mitigate Winner-Take-All Resource Competition

et al. 2022

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The effects of host resource limitations on the function of synthetic gene circuits have gained significant attention over the past years. Hosts, having evolved resource capacities optimal for their own genome, have been repeatedly demonstrated to suffer from the added burden of synthetic genetic programs, which may in return pose deleterious effects on the circuit’s function. Three resource controller archetypes have been proposed previously to mitigate resource distribution problems in dynamic circuits: the local controller, the global controller, and a “negatively competitive” regulatory (NCR) controller that utilizes synthetic competition to combat resource competition. The dynamics of negative feedback forms of these controllers have been previously investigated, and here we extend the analysis of these resource allocation strategies to the incoherent feedforward loop (iFFL) topology. We demonstrate that the three iFFL controllers can attenuate Winner-Take-All resource competition between two bistable switches. We uncover that the parameters associated with the synthetic competition in the NCR iFFL controller are paramount to its increased efficacy over the local controller type, while the global controllers demonstrate to be relatively ineffectual. Interestingly, unlike the negative feedback counterpart topologies, iFFL controllers exhibit a unique coupling of switch activation thresholds which we term the “coactivation threshold shift” effect. Finally, we demonstrate that a nearly fully orthogonal set of bistable switches could be achieved by pairing an NCR controller with an appropriate level of controller resource consumption.

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“…Understanding of how circuits integrate into the native cellular context is important for preventing circuit retroactivity within the model and within the circuit. Circuit retroactivity, where the system does not function as intended due to coupling to other systems within the cell or to the specific cell states, represents a notable challenge to robust circuit design [95,102,103]. Similarly, examples from introduction of synthetic effectors of the RNAi pathway indicate that synthetic components can sequester resources and systemically alter cellular behavior [104][105][106].…”

Section: Trends In Biotechnologymentioning

confidence: 99%

Synthetic gene circuits as tools for drug discovery

Beitz

Oakes

Galloway

2022

Trends in Biotechnology

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Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS). By tracking events and cellular states across multiple length and time scales, circuits will transform how we decipher the causal link between molecular events and phenotypes to improve the selectivity and sensitivity of cell-based assays. Enhancing phenotypic drug discovery with synthetic biologyDevelopment of new drugs requires identification and optimization of functional molecules that often proceeds sequentially through high-throughput discovery screening, hit validation, hit expansion and optimization, and preclinical disease modeling (see Glossary) (Figure 1, Key figure)[1]. Target-based drug discovery (TDD) programs aim to identify drug candidates that modulate a defined biological target that is hypothesized to play a causal role in initiating or sustaining a disease. TDD programs offer precision by directly screening for drug candidate efficacy using purified molecular targets [2]. By contrast, phenotypic drug discovery (PDD) programs aim to identify drug candidates that modulate a physiologically-relevant biological system or cellular signaling pathway and screen for drug efficacy using cell-based assays [2]. Cell-based assays are target-agnostic and provide the opportunity to identify therapeutics that resolve diseaseassociated phenotypic deficits even when the disease etiology remains obscure and targets are undefined. Thus, PDD has recently received renewed interest for its potential to identify first-in-class drugs that act via a novel mechanism of action (MoA) as well as identify drugs for disease with complex or unknown etiology [1][2][3][4]. In order to identify drug candidates that translate to effective therapeutics in the clinic, PDD programs require cell-based assays that faithfully recapitulate and report on disease-relevant processes. To efficiently and effectively screen in phenotypic models, assay development for initial discovery screens must balance throughput with the ability to capture these disease processes [1]. As PDD probes a larger biological space than TDD to identify potential therapeutics, the number of hits from an initial discovery screen in a PDD program may be very large [2]. Following initial discovery screens, hit validation and triage are required to produce a small number of high-confidence candidates to proceed to the resource-intensive stages of hit expansion, compound optimization, and hig...

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Feedforward ribosome control mitigates gene activation burden

Cited by 5 publications

References 114 publications

Context-aware synthetic biology by controller design: Engineering the mammalian cell

Context-aware synthetic biology by controller design: Engineering the mammalian cell

Negatively Competitive Incoherent Feedforward Loops Mitigate Winner-Take-All Resource Competition

Synthetic gene circuits as tools for drug discovery

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