The crystallization of organically capped nanoparticles, unlike the hard-sphere crystallization of atoms, molecules, or conventional colloids, is a "soft process" in which the deformation of organic layers (soft coronae) unavoidably occurs. Despite previous efforts that focused mainly on structures at thermodynamic equilibrium, [1][2][3][4][5][6][7][8][9][10][11] it is not known how soft coronae deform dynamically in this soft-crystallization process. Here, using DNA-capped nanoparticles as a model system, we have probed in real time and in situ the entire drying-mediated soft-crystallization process by synchrotronbased small-angle X-ray scattering (SAXS). Notably, in our DNA-based approach [10,11] the known strategy of programmable crystal formation [12][13][14] is combined with drying-mediated self-assembly. [15] Our dynamic studies demonstrate that our soft crystals have continuously scalable crystalline states with a gradual transition from "wet crystals" to "dry crystals". We have found that the drying-mediated deformation of DNA molecules is elastic in accordance with an entropic spring model, which can also be applied in general to the drying-mediated self-assembly of other organically capped inorganic nanoparticles.We define the softness of the soft-corona/solid-core particle by the dimensionless quantity c = 2 h 0 /d core , where h 0 is the effective height of the corona layer and d core is the diameter of the nanoparticle core (Figure 1). Compared to alkyl-chain-derivatized nanoparticles, [1][2][3][4][5]7] DNA-capped nanoparticles are much softer spheres. Based on our definition, we obtain a typical softness of 0.3-0.8 for alkyl-corona nanoparticles [16] and a softness of 0.6-5.1 for our DNA-corona nanoparticles (these consist of nanoparticles 13 nm in diameter end-grafted with single-stranded DNA (ssDNA) 5 to 90 bases in length). In contrast to the crystallization of colloidal hard spheres (c = 0), the drying-mediated stress that water imparts on the nanoparticles can lead to the deformation of soft-corona nanoparticles (c > 0). This stress is derived from surface tension, which increases with water evaporation. [17] We mapped comprehensively (both temporally and spatially) the crystallization events of different DNAcapped nanoparticles over the entire lifetime of a drying droplet by means of real-time and in situ synchrotron-based SAXS. One-dimensional (1D) SAXS data (structure factor S(q) versus scattering vector q; for details see the Supporting Information) was used to index crystalline lattices and quantitatively determine the nearest-neighbor spacing, D NN . We first showed that nanoparticle supracrystals form for all DNA sequences investigated (see Section 1 in the Supporting Information), regardless of whether the sequences contain Watson-Crick base-pairing regions. However, the crystallization time, t c , at which supracrystals start to form, varies between sequences. We compared time-lapse 2D SAXS images from the entire drying periods for 5'-TGTAC and [*] Dr.

No abstract

In natural environments, microbes are typically non‐dividing and gauge when nutrients permit division. Current models are phenomenological and specific to nutrient‐rich, exponentially growing cells, thus cannot predict the first division under limiting nutrient availability. To assess this regime, we supplied starving Escherichia coli with glucose pulses at increasing frequencies. Real‐time metabolomics and microfluidic single‐cell microscopy revealed unexpected, rapid protein, and nucleic acid synthesis already from minuscule glucose pulses in non‐dividing cells. Additionally, the lag time to first division shortened as pulsing frequency increased. We pinpointed division timing and dependence on nutrient frequency to the changing abundance of the division protein FtsZ. A dynamic, mechanistic model quantitatively relates lag time to FtsZ synthesis from nutrient pulses and FtsZ protease‐dependent degradation. Lag time changed in model‐congruent manners, when we experimentally modulated the synthesis or degradation of FtsZ. Thus, limiting abundance of FtsZ can quantitatively predict timing of the first cell division.

Vaccines based on virus-like particles have proved their success in human health. More than 25 years after the approval of the first vaccine based on this technology, the substantial efforts to expand the range of applications and target diseases are beginning to bear fruit. The incursion of high-throughput screening technologies, combined with new developments in protein engineering and chemical coupling, have accelerated the development of systems capable of producing macrostructures useful for vaccinology, gene delivery, immunotherapy and bionanotechnology. This review summarizes the most recent developments in microbial cell factories and cell-free systems for virus-like particle production and discusses the future impact of this technology in human and animal health.

Changing nutritional conditions challenge microbes and shape their evolutionary optimization. Here, we used real-time metabolomics to investigate the role of glycogen in the dynamic physiological adaptation of Escherichia coli to fluctuating nutrients following carbon starvation. After the depletion of environmental glucose, we found significant metabolic activity remaining, which was linked to rapid utilization of intracellular glycogen. Glycogen was depleted by 80% within minutes of glucose starvation and was similarly replenished within minutes of glucose availability. These fast time scales of glycogen utilization correspond to the short-term benefits that glycogen provided to cells undergoing various physiological transitions. Cells capable of utilizing glycogen exhibited shorter lag times than glycogen mutants when starved between periods of exposure to different carbon sources. The ability to utilize glycogen was also important for the transition between planktonic and biofilm lifestyles and enabled increased glucose uptake during pulses of limited glucose availability. While wild-type and mutant strains exhibited comparable growth rates in steady environments, mutants deficient in glycogen utilization grew more poorly in environments that fluctuated on minute scales between carbon availability and starvation. Taken together, these results highlight an underappreciated role of glycogen in rapidly providing carbon and energy in changing environments, thereby increasing survival and competition capabilities under fluctuating and nutrient-poor conditions. IMPORTANCE Nothing is constant in life, and microbes in particular have to adapt to frequent and rapid environmental changes. Here, we used real-time metabolomics and single-cell imaging to demonstrate that the internal storage polymer glycogen plays a crucial role in such dynamic adaptations. Glycogen is depleted within minutes of glucose starvation and similarly is replenished within minutes of glucose availability. Cells capable of utilizing glycogen exhibited shorter lag times than glycogen mutants when starved between periods of exposure to different carbon sources. While wild-type and mutant strains exhibited comparable growth rates in steady environments, mutants deficient in glycogen utilization grew more poorly in environments that fluctuated on minute scales between carbon availability and starvation. These results highlight an underappreciated role of glycogen in rapidly providing carbon and energy in changing environments, thereby increasing survival and competition capabilities under fluctuating and nutrient-poor conditions.

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant—although lower—level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells’ response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells’ immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via β-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and β-oxidation as important energy providers during acute glucose starvation.

Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits.

The crystallization of organically capped nanoparticles, unlike the hard-sphere crystallization of atoms, molecules, or conventional colloids, is a "soft process" in which the deformation of organic layers (soft coronae) unavoidably occurs. Despite previous efforts that focused mainly on structures at thermodynamic equilibrium, [1][2][3][4][5][6][7][8][9][10][11] it is not known how soft coronae deform dynamically in this soft-crystallization process. Here, using DNA-capped nanoparticles as a model system, we have probed in real time and in situ the entire drying-mediated soft-crystallization process by synchrotronbased small-angle X-ray scattering (SAXS). Notably, in our DNA-based approach [10,11] the known strategy of programmable crystal formation [12][13][14] is combined with drying-mediated self-assembly. [15] Our dynamic studies demonstrate that our soft crystals have continuously scalable crystalline states with a gradual transition from "wet crystals" to "dry crystals". We have found that the drying-mediated deformation of DNA molecules is elastic in accordance with an entropic spring model, which can also be applied in general to the drying-mediated self-assembly of other organically capped inorganic nanoparticles.We define the softness of the soft-corona/solid-core particle by the dimensionless quantity c = 2 h 0 /d core , where h 0 is the effective height of the corona layer and d core is the diameter of the nanoparticle core (Figure 1). Compared to alkyl-chain-derivatized nanoparticles, [1][2][3][4][5]7] DNA-capped nanoparticles are much softer spheres. Based on our definition, we obtain a typical softness of 0.3-0.8 for alkyl-corona nanoparticles [16] and a softness of 0.6-5.1 for our DNA-corona nanoparticles (these consist of nanoparticles 13 nm in diameter end-grafted with single-stranded DNA (ssDNA) 5 to 90 bases in length). In contrast to the crystallization of colloidal hard spheres (c = 0), the drying-mediated stress that water imparts on the nanoparticles can lead to the deformation of soft-corona nanoparticles (c > 0). This stress is derived from surface tension, which increases with water evaporation. [17] We mapped comprehensively (both temporally and spatially) the crystallization events of different DNAcapped nanoparticles over the entire lifetime of a drying droplet by means of real-time and in situ synchrotron-based SAXS. One-dimensional (1D) SAXS data (structure factor S(q) versus scattering vector q; for details see the Supporting Information) was used to index crystalline lattices and quantitatively determine the nearest-neighbor spacing, D NN . We first showed that nanoparticle supracrystals form for all DNA sequences investigated (see Section 1 in the Supporting Information), regardless of whether the sequences contain Watson-Crick base-pairing regions. However, the crystallization time, t c , at which supracrystals start to form, varies between sequences. We compared time-lapse 2D SAXS images from the entire drying periods for 5'-TGTAC and [*] Dr.