A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

Abstract Background Plasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida. Conclusion As a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering

Band structure engineered layered metals for low-loss plasmonics

Plasmonics currently faces the problem of seemingly inevitable optical losses occurring in the metallic components that challenges the implementation of essentially any application. In this work we show that Ohmic losses are reduced in certain layered metals, such as the transition metal dichalcogenide TaS$_2$, due to an extraordinarily small density of states for scattering in the near-IR originating from their special electronic band structure. Based on this observation we propose a new class of band structure engineered van der Waals layered metals composed of hexagonal transition metal chalcogenide-halide layers with greatly suppressed intrinsic losses. Using first-principles calculations we show that the suppression of optical losses lead to improved performance for thin film waveguiding and transformation optics

Improving the decision-making basis by strengthening the risk assessments of unexploded ordnance and explosive remnants of war

For many countries, the legacy of armed conflict in the form of unexploded ordnance has a severe impact on society and daily life, as millions of tonnes of explosive remnants of war represent a grave threat to both the environment and societal safety and security. Recent and dramatic changes in the security situation in Europe sadly demonstrate that explosive remnants of war are not, however, only a thing of the past. This makes it especially relevant to evaluate how we assess and manage this risk today and how, if possible, this practice could be improved. In the present paper, we will outline some of the particularities that differentiate risk assessments of unexploded ordnance from other, more familiar, risks and discuss whether the current methodology can be considered relevant and appropriate. We find that the different risk assessment methodologies generally in use today, as described in applicable guidelines and regulations, are principally unsuitable for this use and, in addition, sometimes also ambiguous, inconsistent and incompatible. In particular, we find that any model based on a risk assessment that does not include an evaluation of background knowledge and associated uncertainties cannot be regarded as an optimal or appropriate risk assessment tool, when assessing a risk typically characterized by high complexity and uncertainty. The conclusion of this investigation is that the current risk assessment methodology for assessing risks related to unexploded ordnance and explosive remnants of war urgently needs to be revised, in order to improve the decision-making basis.publishedVersio