Routinizing the engineering of synthetic cells requires specifying beforehand how many of each molecule are needed. Physics-based tools for estimating desired molecular abundances in whole-cell ...synthetic biology are missing. Here, we use a colloidal dynamics simulator to make predictions for how tRNA abundances impact protein synthesis rates. We use rational design and direct RNA synthesis to make 21 synthetic tRNA surrogates from scratch. We use evolutionary algorithms within a computer aided design framework to engineer translation systems predicted to work faster or slower depending on tRNA abundance differences. We build and test the so-specified synthetic systems and find qualitative agreement between expected and observed systems. First principles modeling combined with bottom-up experiments can help molecular-to-cellular scale synthetic biology realize design-build-work frameworks that transcend tinker-and-test.
Faster-growing cells must synthesize proteins more quickly. Increased ribosome abundance only partly accounts for increases in total protein synthesis rates. The productivity of individual ribosomes ...must increase too, almost doubling by an unknown mechanism. Prior models point to diffusive transport as a limiting factor but raise a paradox: faster-growing cells are more crowded, yet crowding slows diffusion. We suspected that physical crowding, transport, and stoichiometry, considered together, might reveal a more nuanced explanation. To investigate, we built a first-principles physics-based model of Escherichia coli cytoplasm in which Brownian motion and diffusion arise directly from physical interactions between individual molecules of finite size, density, and physiological abundance. Using our microscopically detailed model, we predicted that physical transport of individual ternary complexes accounts for ~80% of translation elongation latency. We also found that volumetric crowding increases during faster growth even as cytoplasmic mass density remains relatively constant. Despite slowed diffusion, we predicted that improved proximity between ternary complexes and ribosomes wins out, illustrating a simple physics-based mechanism for how individual elongating ribosomes become more productive. We speculate that crowding imposes a physical limit on growth rate and undergirds cellular behavior more broadly. Unfitted colloidal-scale modeling offers systems biology a complementary "physics engine" for exploring how cellular-scale behaviors arise from physical transport and reactions among individual molecules.
Ribosomes are the factories in cells that synthesize proteins. When cells grow faster, there are not enough ribosomes to keep up with the demand for faster protein synthesis without individual ribosomes becoming more productive. Yet, faster-growing cells are more crowded, seemingly making it harder for each ribosome to do its work. Our computational model of the physics of translation elongation reveals the underlying mechanism for how individual ribosomes become more productive: proximity and stoichiometry of translation molecules overcome crowding. Our model also suggests a universal physical limitation of cell growth rates.
Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly ...features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.
Building cells from scratch requires understanding how molecules -- the building blocks of cells -- move and interact to produce behavior. To better address this challenge I apply colloidal physics ...-- how microscopic particles behave in liquids -- to cell biology, leveraging protein synthesis as a model process. Specifically, I develop a modeling framework for representing the physical motion and chemical reactions of individual molecules in the full context of crowded cytoplasm, showing how individual tRNA and ribosomes diffuse and interact to enable protein synthesis. Using my framework, I elucidate a putative mechanism underlying how ribosomes become more productive in faster growing cells, an experimental observation that has remained unexplained for 50 years. Specifically, I show that the nucleoid-excluded cytoplasm, where translation primarily occurs, becomes three times more packed with molecules in faster growing cells. While increased packing slows molecular movement down, molecules also become closer together, a general phenomenon that enables matching tRNA and ribosomes to find each other more quickly and thus speeds up protein synthesis. I then explore the expected properties of cytoplasm for hypothetical cells that grow faster than ever observed. I predict that further increases in crowding would provide diminishing returns for or hinder protein synthesis, suggesting a physical limit on growth rate. Finally, I extend colloidal physics modeling directly to the design of synthetic cells by calculating how the relative abundances of tRNA can be engineered to provide optimal protein synthesis strategies for both wild-type and codon-reduced genomes. Engineered tRNA abundances are predicted to enable up to ~20% faster and ~50% slower protein synthesis compared to natural abundances. Colloidal physics modeling and engineering can be expanded across cell types and processes, supporting the general design and construction of cells.
Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly ...features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.
Postamputation phantom pain is notoriously persistent with few validated treatments. Cryoneurolysis involves the application of low temperatures to reversibly ablate peripheral nerves. The authors ...tested the hypothesis that a single cryoneurolysis treatment would decrease phantom pain 4 months later.
The authors enrolled patients with a lower-limb amputation and established phantom pain. Each received a single-injection femoral and sciatic nerve block with lidocaine and was subsequently randomized to receive either ultrasound-guided percutaneous cryoneurolysis or sham treatment at these same locations. The primary outcome was the change in average phantom pain intensity between baseline and 4 months as measured with a numeric rating scale (0 to 10), after which an optional crossover treatment was offered. Investigators, participants, and clinical staff were masked to treatment group assignment with the exception of the treating physician performing the cryoneurolysis, who had no subsequent participant interaction.
Pretreatment phantom pain scores were similar in both groups, with a median quartiles of 5.0 4.0, 6.0 for active treatment and 5.0 4.0, 7.0 for sham. After 4 months, pain intensity decreased by 0.5 -0.5, 3.0 in patients given cryoneurolysis (n = 71) versus 0 0, 3 in patients given sham (n = 73), with an estimated difference (95% CI) of -0.1 (-1.0 to 0.7), P = 0.759. Following their statistical gatekeeping protocol, the authors did not make inferences or draw conclusions on secondary endpoints. One serious adverse event occurred after a protocol deviation in which a femoral nerve cryolesion was induced just below the inguinal ligament-instead of the sensory-only saphenous nerve-which resulted in quadriceps weakness, and possibly a fall and clavicle fracture.
Percutaneous cryoneurolysis did not decrease chronic lower extremity phantom limb pain 4 months after treatment. However, these results were based upon the authors' specific study protocol, and since the optimal cryoneurolysis treatment parameters such as freeze duration and anatomic treatment location remain unknown, further research is warranted.
The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that ...this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.
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