From Electronic Sequence to Purified Protein Using Automated Gene Synthesis and In Vitro Transcription/Translation.

De novo gene synthesis is the state-of-the-art method used to obtain genetic material adapted to the requirements of the host organism and a cornerstone for modern synthetic biology. Yet, little progress has been made regarding downstream processes of protein production from synthetic genetic material. The production of recombinant proteins traditionally requires extensive preparatory work including gene amplification, cloning, sequencing, transformation or transfection of the expression host, cultivation of living cells, and purification of the overexpressed protein. In this work we describe a fast and automated workflow for cell-free production of proteins starting from an electronic protein sequence or accession number. PRESTO (protein expression starting from oligonucleotides) seamlessly combines a tailored in silico sequence optimization with the assembly of short oligonucleotides into synthetic linear DNA expression cassettes, mammalian in vitro transcription/translation, and protein purification thereof. Integrated on a small liquid handling system it provides a hands-free high throughput source for functional synthetic proteins within 1 day.

Time-resolved coherent X-ray diffraction imaging of surface acoustic waves.

Time-resolved coherent X-ray diffraction experiments of standing surface acoustic waves, illuminated under grazing incidence by a nanofocused synchrotron beam, are reported. The data have been recorded in stroboscopic mode at controlled and varied phase between the acoustic frequency generator and the synchrotron bunch train. At each time delay (phase angle), the coherent far-field diffraction pattern in the small-angle regime is inverted by an iterative algorithm to yield the local instantaneous surface height profile along the optical axis. The results show that periodic nanoscale dynamics can be imaged at high temporal resolution in the range of 50 ps (pulse length).

Pulse-resolved multi-photon X-ray detection at 31 MHz based on a quadrant avalanche photodiode.

The technical realisation and the commissioning experiments of a high-speed X-ray detector based on a quadrant avalanche silicon photodiode and high-speed digitizers are described. The development is driven by the need for X-ray detectors dedicated to time-resolved diffraction and imaging experiments, ideally requiring pulse-resolved data processing at the synchrotron bunch repetition rate. By a novel multi-photon detection scheme, the exact number of X-ray photons within each X-ray pulse can be recorded. Commissioning experiments at beamlines P08 and P10 of the storage ring PETRA III, at DESY, Hamburg, Germany, have been used to validate the pulse-wise multi-photon counting scheme at bunch frequencies ≥ 31 MHz, enabling pulse-by-pulse readout during the PETRA III 240-bunch mode with single-photon detection capability. An X-ray flux of ≥ 3.7 × 10(9) photons s(-1) can be detected while still resolving individual photons at low count rates.

Acyl-chain correlation in membrane fusion intermediates: x-ray diffraction from the rhombohedral lipid phase.

We have studied the acyl-chain conformation in stalk phases of model membranes by x-ray diffraction from oriented samples. As an equilibrium lipid phase induced by dehydration, the stalk or rhombohedral phase exhibits lipidic passages (stalks) between adjacent bilayers, representing a presumed intermediate state in membrane fusion. From the detailed analysis of the acyl-chain correlation peak, we deduce the structural parameters of the acyl-chain fluid above, at, and below the transition from the lamellar to rhombohedral state, at the molecular level.

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition.

We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 ± 0.5 Å for the investigated lipid compositions.