Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution

Dickinson, Bryan C.; Leconte, Aaron M.; Allen, Benjamin; Esvelt, Kevin M.; Liu, David R.

doi:10.1073/pnas.1220670110

Cited by 99 publications

(125 citation statements)

References 35 publications

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“…We chose to deploy T7 RNAP split at position 179 because, 1) the N-terminal half is small, 2) structural data indicates this position is solvent exposed and removed from the DNA-binding face of the protein, and 3) mutations that influence DNA promoter specificity are C-terminal to this position 26,30,31 . First, we validated that the two RNAP halves spontaneously assemble using an Escherichia coli ( E. coli ) luciferase reporter system (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Such a protein engineering problem presents substantial challenges, which can in principle be overcome by molecular evolution. Therefore, we chose to deploy PACE, a rapid evolution system 35 , which has been used to evolve RNAP promoter specificity, protein-DNA interactions, protease activities, and protein-protein interactions 3,30,35–40 . Briefly, PACE involves providing an evolving gene of interest to M13 bacteriophage, linking the life cycle of the phage to a desired activity of interest to be evolved in the target gene, and then propagating the virus until the activity evolves.…”

Section: Resultsmentioning

confidence: 99%

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Evolution of a split RNA polymerase as a versatile biosensor platform

2017

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Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies often suffer from a lack of signal-to-noise, requirements for extensive optimization for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting “activity-responsive RNAP” (AR) system to create light and small molecule activated biosensors, demonstrating the “plug-and-play” nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Evolution of a split RNA polymerase as a versatile biosensor platform

2017

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“…One way to test this prediction is to reconstruct the order of substitutions in which evolution proceeded and then to test whether accumulation of the substitutions in a different order is possible [45,[76][77][78][79][80][81][82]. A variation of this test was performed recently [82] for a nucleoprotein in the human H3N2 influenza virus.…”

Section: Order Of Substitutions In Evolutionmentioning

confidence: 99%

Topological features of rugged fitness landscapes in sequence space

Kondrashov

2015

Trends in Genetics

107

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“…In the absence of epistasis, a mutation has the same effect regardless of its context and therefore regardless of any prior history or subsequent evolution. By contrast, in the presence of epistasis, each substitution may be contingent on the entire prior history of the protein, and it may constrain all subsequent evolution.The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1, 8, 25-34), nor have the concepts of contingency (3,4,9,12,(35)(36)(37)(38) and, more recently, entrenchment (18,39,40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1-9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18,(41)(42)(43)(44).…”

mentioning

confidence: 99%

“…The potential for epistasis to play an important role in evolution, including protein evolution, has not been overlooked by researchers (1,8,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), nor have the concepts of contingency (3,4,9,12,(35)(36)(37)(38) and, more recently, entrenchment (18,39,40). However, most studies have addressed the role of epistasis in the context of adaptive evolution (1-9, 27, 30, 31, 36, 38), whereas the consequences of epistasis under purifying selection have received less attention (18,(41)(42)(43)(44).…”

mentioning

confidence: 99%

Contingency and entrenchment in protein evolution under purifying selection

Shah

McCandlish

Plotkin

2015

Proc. Natl. Acad. Sci. U.S.A.

182

236

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The phenotypic effect of an allele at one genetic site may depend on alleles at other sites, a phenomenon known as epistasis. Epistasis can profoundly influence the process of evolution in populations and shape the patterns of protein divergence across species. Whereas epistasis between adaptive substitutions has been studied extensively, relatively little is known about epistasis under purifying selection. Here we use computational models of thermodynamic stability in a ligand-binding protein to explore the structure of epistasis in simulations of protein sequence evolution. Even though the predicted effects on stability of random mutations are almost completely additive, the mutations that fix under purifying selection are enriched for epistasis. In particular, the mutations that fix are contingent on previous substitutions: Although nearly neutral at their time of fixation, these mutations would be deleterious in the absence of preceding substitutions. Conversely, substitutions under purifying selection are subsequently entrenched by epistasis with later substitutions: They become increasingly deleterious to revert over time. Our results imply that, even under purifying selection, protein sequence evolution is often contingent on history and so it cannot be predicted by the phenotypic effects of mutations assayed in the ancestral background.intragenic epistasis | coevolution | near neutrality | protein stability W hether a heritable mutation is advantageous or deleterious to an organism often depends on the evolutionary history of the population. A mutation that is beneficial at the time of its introduction may confer its beneficial effect only in the presence of other potentiating or permissive mutations (1-9). Thus, the fate of a mutation arising in a population may be contingent on previous mutations (10-13). Conversely, once a mutation has fixed in a population, the mutation becomes part of the genetic background onto which subsequent modifications are introduced. Because the beneficial effects of the subsequent modifications may depend on the focal mutation, as time passes reversion of the focal mutation may become increasingly deleterious, leading to a type of evolutionary conservatism, or entrenchment (14-18).In the context of protein evolution, the effects of contingency and entrenchment are most easily studied by considering a sequence of single amino acid changes (19) that extends both forward and backward in time from some focal substitution. To assess the roles of contingency and entrenchment we can study the degree to which each focal substitution was facilitated by previous substitutions, and the degree to which the focal substitution influences the subsequent course of evolution (Fig. 1A).Dependencies within a sequence of substitutions are closely connected to the concept of epistasis-that is, the idea that the phenotypic effect of a mutation at a particular genetic site may depend on the genetic background in which it arises (20-24). In the absence of epistasis, a mutation has the same effect ...

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Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution

Cited by 99 publications

References 35 publications

Evolution of a split RNA polymerase as a versatile biosensor platform

Evolution of a split RNA polymerase as a versatile biosensor platform

Topological features of rugged fitness landscapes in sequence space

Contingency and entrenchment in protein evolution under purifying selection

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