Resource-aware construct design in mammalian cells

Blasi, Roberto Di; Pisani, Mara; Tedeschi, Fabiana; Marbiah, Masue; Polizzi, Karen M.; Furini, Simone; Siciliano, Velia; Ceroni, Francesca

doi:10.1038/s41467-023-39252-4

Cited by 13 publications

(15 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Capacity monitors have been previously adopted to quantify and visualise gene expression resource competition in bacteria and mammalian cells 34,35,[45][46][47][48] . Monitors are fluorescent expression cassettes that function as proxy for the available gene expression capacity within a cellular host.…”

Section: Competition For Degradation Resources Impacts Mammalian Expr...mentioning

confidence: 99%

“…Mather et al expanded the concept of competition for degradation resources in bacteria by characterising crosstalk involving other bacterial proteases such as ClpAP and Lon 32,33 . Previous work on resource competition in mammalian cells characterised bottlenecks in the transcriptional, translational and secretory machinery during transient gene expression [34][35][36][37][38] . However, considerations of how limitations in protein degradation resources impact heterologous gene expression have not been reported for these systems, yet 39 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Degradation Bottlenecks and Resource Competition in Transiently and Stably Engineered Mammalian Cells

Gabrielli,

Di Blasi,

Kontoravdi

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Degradation tags, otherwise known as degrons, are portable sequences that can be used to alter protein stability. Here, we report that degron-tagged proteins compete for cellular degradation resources in engineered mammalian cells leading to coupling of the degradation rates of otherwise independently expressed proteins when constitutively targeted human degrons are adopted. By adopting inducible bacterial and plant degrons we also highlight how orthogonality and uncoupling of synthetic construct degradation from the native machinery can be achieved. We show the effect of this competition to be dependent on the context of the degrons where C-terminal degradation appears to impact competition the most across our tested settings. We then build a genomically integrated capacity monitor tagged with different degrons and confirm resource competition between genomic and transiently expressed DNA constructs. This work expands the characterisation of resource competition in engineered mammalian cells to degradation also including integrated systems, providing a framework for the optimisation of heterologous expression systems to advance applications in fundamental and applied biological research.

show abstract

Section: Competition For Degradation Resources Impacts Mammalian Expr...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Degradation Bottlenecks and Resource Competition in Transiently and Stably Engineered Mammalian Cells

Gabrielli,

Di Blasi,

Kontoravdi

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…One fundamental challenge in synthetic gene circuit design is posed by resource competition, which causes unintended interplay between circuit modules [1][2][3][4][5][6][7]. Specifically, this interplay arises from the competition for limited cellular resources, such as RNA polymerases (RNAPs), ribosomes, and transcription factors, among the exogenous genes within the same host cell [8][9][10][10][11][12]. This undesired coupling not only alters the deterministic behavior of synthetic gene circuits dramatically but also diminishes their robustness by inducing significant fluctuations in gene expression.…”

Section: Introductionmentioning

confidence: 99%

Noise Reduction in Resource-Coupled Multi-Module Gene Circuits through Antithetic Feedback Control

Chakravarty,

Zhang,

Tian

2024

Preprint

View full text Add to dashboard Cite

Gene circuits within the same host cell often experience coupling, stemming from the competition for limited resources during transcriptional and translational processes. This resource competition introduces an additional layer of noise to gene expression. Here we present three multi-module antithetic control strategies: negatively competitive regulation (NCR) controller, alongside local and global controllers, aimed at reducing the gene expression noise within the context of resource competition. Through stochastic simulations and fluctuation-dissipation theorem (FDT) analysis, our findings highlight the superior performance of the NCR antithetic controller in reducing noise levels. Our research provides an effective control strategy for attenuating resource-driven noise and offers insight into the development of robust gene circuits.

show abstract

“…However, despite their potential, prototyping with clinically relevant cells, in particular human pluripotent stem cells (hPSC) has been severely hampered by several major barriers. These include the immunogenicity of synthetic components, unpredictable context effects, resource overload, and epigenetic silencing [30][31][32][33] . An overarching challenge-preventing timely solution development on all these fronts--is the paucity of engineering tools for hPSCs and the hitherto unavoidable slow pace of design iteration.…”

Section: Introductionmentioning

confidence: 99%

FAST-STEM: A human pluripotent stem cell engineering toolkit for rapid design-build-test-learn development of human cell-based therapeutic devices

Rosenstein,

Sambathkumar,

Murareanu

et al. 2024

Preprint

View full text Add to dashboard Cite

Very recent clinical advances in stem cell derived tissue replacement and gene therapy, in addition to the rise of artificial intelligence aided scientific discovery, have placed the possibility of sophisticated human cell based therapies firmly within reach. However, development of such cells and testing of their engineered gene circuit components, has proven highly challenging, due to the need for generating stable cell lines for each design, build, test, learn engineering cycle. Current approaches to generating stable human induced pluripotent stem cell (hiPSC) lines are highly time consuming and suffer from lack of control, poor integration efficiency, and limited functionality. Validation in clinically relevant stem cell derived tissues is also broadly lacking. Such drawbacks are prohibitive to repeatably conducting cutting edge stem cell engineering with broad application within a realistic timeframe, and will not scale with the future of regenerative medicine. We have developed FASTSTEM (Facile Accelerated Stem cell Transgene integration with SynBio Tunable Engineering Modes), a hiPSC engineering platform that drastically reduces the time to generate differentiation ready stem cell lines from several weeks to 5 days, exhibiting a ~612-fold improvement in transgene integration rate over previous methodologies. Additional FAST-STEM innovations include: (i) rapid and highly efficient transgene integration; (ii) copy number control; (iii) simultaneous or consecutive integration of multiple gene cassettes; (iv) library screen capability. In addition to this unique functional versatility, platform transportability and broad use case for stem cell engineering was confirmed by differentiation into eight different cell types across nine different laboratories. This platform dramatically lowers the bar for integration of synthetic biology with regenerative medicine, enabling experiments which were previously deemed logistically impossible, thus paving the way for sophisticated human cell device development.

show abstract

Resource-aware construct design in mammalian cells

Cited by 13 publications

References 48 publications

Degradation Bottlenecks and Resource Competition in Transiently and Stably Engineered Mammalian Cells

Degradation Bottlenecks and Resource Competition in Transiently and Stably Engineered Mammalian Cells

Noise Reduction in Resource-Coupled Multi-Module Gene Circuits through Antithetic Feedback Control

FAST-STEM: A human pluripotent stem cell engineering toolkit for rapid design-build-test-learn development of human cell-based therapeutic devices

Contact Info

Product

Resources

About