In situ Rb-Sr dating by collision cell, multicollection inductively-coupled plasma mass-spectrometry with pre-cell mass-filter, (CC-MC-ICPMS/MS)

We document the utility for in situ Rb–Sr dating of a one-of-a-kind tribrid mass spectrometer, ‘Proteus’, coupled to a UV laser ablation system. Proteus combines quadrupole mass-filter, collision cell and sector magnet with a multicollection inductively-coupled plasma mass spectrometer (CC-MC-ICPMS/MS). Compared to commercial, single collector, tribrid inductively-coupled plasma mass spectrometers (CC-ICPMS/MS) Proteus has enhanced ion transmission and offers simultaneous collection of all Sr isotopes using an array of Faraday cups. These features yield improved precision in measured (87)Sr/(86)Sr ratios, for a given mass of Sr analysed, approximately a factor of 25 in comparison to the Thermo Scientific™ iCAP TQ™ operated under similar conditions. Using SF(6) as a reaction gas on Proteus, measurements of Rb-doped NIST SRM (standard reference material) 987 solutions, with Rb/Sr ratios from 0.01–100, yield (87)Sr/(86)Sr that are indistinguishable from un-doped NIST SRM 987, demonstrating quantitative ‘chemical resolution’ of Rb from Sr. We highlight the importance of mass-filtering before the collision cell for laser ablation (87)Sr/(86)Sr analysis, using an in-house feldspar standard and a range of glass reference materials. By transmitting only those ions with mass-to-charge ratios 82–92 u/e into the collision cell, we achieve accurate (87)Sr/(86)Sr measurements without any corrections for atomic or polyatomic isobaric interferences. Without the pre-cell mass-filtering, measured in situ(87)Sr/(86)Sr ratios are inaccurate. Combining in situ measurements of Rb/Sr and radiogenic Sr isotope ratios we obtain mineral isochrons. We utilise a sample from the well-dated Dartmoor granite (285 ± 1 Ma) as a calibrant for our in situ ages and, using the same conditions, produce accurate Rb–Sr isochron ages for samples of the Fish Canyon tuff (28 ± 2 Ma) and Shap granite pluton (397 ± 1 Ma). Analysing the same Dartmoor granite sample using identical laser conditions and number of spot analyses using the Thermo Scientific™ iCAP TQ™ yielded an isochron slope 5× less precise than Proteus. We use an uncertainty model to illustrate the advantage of using Proteus over single collector CC-ICPMS/MS for in situ Rb–Sr dating. The results of this model show that the improvement is most marked for samples that have low Rb/Sr (<10) or are young (<100 Ma). We also report the first example of an in situ, internal Rb–Sr isochron from a single potassium-feldspar grain. Using a sample from the Shap granite, we obtained accurate age and initial (87)Sr/(86)Sr with 95% confidence intervals of ±1.5% and ±0.03% respectively. Such capabilities offer new opportunities in geochronological studies

A new dynamic module for in-situ nanomechanical testing at high strain rate

In-situ nanomechanical testing is commonly used to probe surface mechanical properties of bulk materials or thin films, like hardness, Young’s modulus, Yield stress…Actually most of the instruments can measure these properties only statically, i.e. a low frequency, leading to property measurement only at low strain rate (usually 10-1s-1 by nanoindentation). This is mainly caused by the low resonance frequency of the system, preventing making tests at higher speed. Performing high dynamic measurements could bring new information on materials properties like deformation mechanism at high strain rate, or high dynamic fatigue properties. A new high dynamic module usable for in-situ mechanical testing has been developed. It is composed of a small piezotube attached directly behind the tip. Because of the small dimensions of the module, his resonance frequency is very high (higher than 50kHz) in comparison to classical nanomechanical testers, permitting to perform and measure precisely the signals at very high frequency. Moreover, it can be used as a sensor and as an actuator, in x, y and z directions which gives to this module a very large range of measurements. Firstly, the characteristics, the performances and the limits of the new high dynamic module will be presented. Secondly some indentations experiments performed at high strain rate on nanocrystalline nickel with the in-situ nanomechanical tester (Alemnis Gmbh) equipped with the high dynamic will be presented and discussed (Fig. 1). Finally, some micropillar compression at high strain rate on the same material will be described and discussed

A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae

Despite the extensive use of&nbsp;Saccharomyces cerevisiae&nbsp;as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation&nbsp;and solubility tags.&nbsp;We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production

Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in <i>S. cerevisiae</i>

Abstract Background Beyond pathway engineering, the metabolic state of the production host is critical in maintaining the efficiency of cellular production. The biotechnologically important yeast Saccharomyces cerevisiae adjusts its energy metabolism based on the availability of oxygen and carbon sources. This transition between respiratory and non-respiratory metabolic state is accompanied by substantial modifications of central carbon metabolism, which impact the efficiency of metabolic pathways and the corresponding final product titers. Non-ribosomal peptide synthetases (NRPS) are an important class of biocatalysts that provide access to a wide array of secondary metabolites. Indigoidine, a blue pigment, is a representative NRP that is valuable by itself as a renewably produced pigment. Results Saccharomyces cerevisiae was engineered to express a bacterial NRPS that converts glutamine to indigoidine. We characterize carbon source use and production dynamics, and demonstrate that indigoidine is solely produced during respiratory cell growth. Production of indigoidine is abolished during non-respiratory growth even under aerobic conditions. By promoting respiratory conditions via controlled feeding, we scaled the production to a 2 L bioreactor scale, reaching a maximum titer of 980 mg/L. Conclusions This study represents the first use of the Streptomyces lavendulae NRPS (BpsA) in a fungal host and its scale-up. The final product indigoidine is linked to the activity of the TCA cycle and serves as a reporter for the respiratory state of S. cerevisiae. Our approach can be broadly applied to investigate diversion of flux from central carbon metabolism for NRPS and other heterologous pathway engineering, or to follow a population switch between respiratory and non-respiratory modes