Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS)

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD(+)-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.This research was funded by the Engineering and Physical Sciences Research Council (studentship to RH and an Impact Acceleration Account Partnership Development Award), the Biological and Biotechnological Research Council (BBSRC) and Johnson Matthey. SE and MF were supported by postdoctoral Marie-Curie fellowships

Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS)

This is the final version. Available from National Academy of Sciences via the DOI in this recordUltrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydro-genase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12°C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.Biotechnology and Biological Sciences Research CouncilEuropean Research CouncilEngineering and Physical Sciences Research CouncilEuropean Commissio

Robust self-scheduling of a price-maker energy storage facility in the New York electricity market

Recent progress in energy storage raises the possibility of creating large-scale storage facilities at lower costs. This may bring economic opportunities for storage operators, especially via energy arbitrage. However, storage operation in the market could have a noticeable impact on electricity prices. This work aims at evaluating jointly the potential operating profit for a price-maker storage facility and its impact on the electricity prices in the New York state market. Based on historical data, lower and upper bounds on the supply curve of the market are constructed. These bounds are used as an input for the robust self-scheduling problem of a price-maker storage facility. Our computational experiments show that the robust strategies thus obtained allow to reduce significantly the loss exposure while maintaining reasonably high expected profits

Deterministic ratchets: route to diffusive transport

The rectification efficiency of an underdamped ratchet operated in the adiabatic regime increases according to a scaling current-amplitude curve as the damping constant approaches a critical threshold; below threshold the rectified signal becomes extremely irregular and eventually its time average drops to zero. Periodic (locked) and diffusive (fully chaotic) trajectories coexist on fine tuning the amplitude of the input signal. The transition from regular to chaotic transport in noiseless ratchets is studied numerically.Comment: 9 pages, 5 figures, to be published in Phys. Rev.

Physics at a 100 TeV pp collider: Higgs and EW symmetry breaking studies

This report summarises the physics opportunities for the study of Higgs bosons and the dynamics of electroweak symmetry breaking at the 100 TeV pp collider.Comment: 187 pages, 94 figures. Chapter 2 of the "Physics at the FCC-hh" Repor

Decoherence and thermalization dynamics of a quantum oscillator

We introduce the quantitative measures characterizing the rates of decoherence and thermalization of quantum systems. We study the time evolution of these measures in the case of a quantum harmonic oscillator whose relaxation is described in the framework of the standard master equation, for various initial states (coherent, `cat', squeezed and number). We establish the conditions under which the true decoherence measure can be approximated by the linear entropy $1-{Tr}\hat\rho^2$. We show that at low temperatures and for highly excited initial states the decoherence process consists of three distinct stages with quite different time scales. In particular, the `cat' states preserve 50% of the initial coherence for a long time interval which increases logarithmically with increase of the initial energy.Comment: 24 pages, LaTex, 8 ps figures, accepted for publication in J. Opt.

Chaos or Noise - Difficulties of a Distinction

In experiments, the dynamical behavior of systems is reflected in time series. Due to the finiteness of the observational data set it is not possible to reconstruct the invariant measure up to arbitrary fine resolution and arbitrary high embedding dimension. These restrictions limit our ability to distinguish between signals generated by different systems, such as regular, chaotic or stochastic ones, when analyzed from a time series point of view. We propose to classify the signal behavior, without referring to any specific model, as stochastic or deterministic on a certain scale of the resolution $\epsilon$, according to the dependence of the $(\epsilon,\tau)$-entropy, $h(\epsilon, \tau)$, and of the finite size Lyapunov exponent, $\lambda(\epsilon)$, on $\epsilon$.Comment: 24 pages RevTeX, 9 eps figures included, two references added, minor corrections, one section has been split in two (submitted to PRE