Mutually Orthogonal DNA Replication Systems <i>In Vivo</i>

Arzumanyan, Garri A.; Gabriel, Kristin; Ravikumar, Arjun; Javanpour, Alex A.; Liu, Chang C.

doi:10.1021/acssynbio.8b00195

Cited by 29 publications

(33 citation statements)

References 25 publications

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“…Another method is to manipulate the size of resource pools 44 , 45 . Orthogonal ribosome pools or orthogonal DNA replication systems have been used to alleviate the effect of resource competition and gene coupling 46 – 48 . Insulation-based engineering strategies have shown to improve the resolution of the genetic circuit 49 .…”

Section: Discussionmentioning

confidence: 99%

Topology-dependent interference of synthetic gene circuit function by growth feedback

Zhang

Melendez-Alvarez

et al. 2020

Nat Chem Biol

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Growth-mediated feedback between synthetic gene circuits and host organisms leads to diverse emerged behaviors, including growth bistability and enhanced ultrasensitivity. However, the range of possible impacts of growth feedback on gene circuits remains underexplored. Here, we mathematically and experimentally demonstrated that growth feedback affects the functions of memory circuits in a network topology-dependent way. Specifically, the memory of the self-activation switch is quickly lost due to the growth-mediated dilution of the circuit products. Decoupling of growth feedback reveals its memory, manifested by its hysteresis property across a broad range of inducer concentration. On the contrary, the toggle switch is more refractory to growth-mediated dilution and can retrieve its memory after the fast-growth phase. The underlying principle lies in the different dependence of active and repressive regulations in these circuits on the growth-mediated dilution. Our results unveil the topology-dependent mechanism on how growth-mediated feedback influences the behaviors of gene circuits.

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Section: Discussionmentioning

confidence: 99%

Topology-dependent interference of synthetic gene circuit function by growth feedback

Zhang

Melendez-Alvarez

et al. 2020

Nat Chem Biol

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“…All of the accessory components required for replication and transcription are encoded on p2. TP-DNAP1 does not replicate p2 and TP-DNAP2 does not replicate p1 (Arzumanyan et al, 2018), meaning that the high error rate of p1 replication does not affect p2-encoded genes and the low error-rate of p2 replication does not compete with p1 mutagenesis. ORFs with unknown function are indicated.…”

Section: Supplementary Materialsmentioning

confidence: 99%

Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds

Ravikumar

Arzumanyan

Obadi

et al. 2018

Cell

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207

202

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Summary Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation. However, experimental strategies for directed evolution are notoriously laborintensive and low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. We report OrthoRep, an orthogonal DNA polymerase-plasmid pair in yeast that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding the error threshold of genomic replication that causes singlegeneration extinction. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly straightforward and scalable process. Using OrthoRep, we evolved drug-resistant malarial DHFRs in 90 independent replicates. We uncovered a more complex fitness landscape than previously realized, including common adaptive trajectories constrained by epistasis, rare outcomes that avoid a frequent early adaptive mutation, and a suboptimal fitness peak that occasionally traps evolving populations. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.

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“…Several approaches have been proposed to counteract the effects of resource competition, either by finetuning the parameters in the gene circuit 5,7 or manipulating the size of the orthogonal resource pools [31][32][33][34] . Additionally, a burden-driven negative feedback loop was implemented to control gene expression by monitoring the cellular burden [35][36][37] .…”

Section: Resource Competition Is Commonplace At Various Levels Of Regmentioning

confidence: 99%

Winner-Takes-All Resource Competition Redirects Cascading Cell Fate Transitions

Zhang

Goetz

Melendez-Alvarez

et al. 2020

Preprint

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Failure of modularity remains a significant challenge for synthetic gene circuits assembled with tested modules as they often do not function as expected. Competition over shared limited gene expression resources is a crucial underlying reason. Here, we first built a synthetic cascading bistable switches (Syn-CBS) circuit in a single strain with two coupled self-activation modules to achieve two successive cell fate transitions. Interestingly, we found that the in vivo transition path was redirected as the activation of one switch always prevailed against the other instead of the theoretically expected coactivation. This qualitatively different type of resource competition between the two modules follows a 'winner-takes-all' rule, where the winner is determined by the relative connection strength between the modules. To decouple the resource competition, we constructed a two-strain circuit, which achieved successive activation and stable coactivation of the two switches.We unveiled a nonlinear resource competition within synthetic gene circuits and provided a division of labor strategy to minimize unfavorable effects.

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Mutually Orthogonal DNA Replication Systems In Vivo

Cited by 29 publications

References 25 publications

Topology-dependent interference of synthetic gene circuit function by growth feedback

Topology-dependent interference of synthetic gene circuit function by growth feedback

Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds

Winner-Takes-All Resource Competition Redirects Cascading Cell Fate Transitions

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