Addicting Escherichia coli to New-to-Nature Reactions

Biocontainment is an essential feature when deploying genetically modified organisms (GMOs) in open system applications, as variants escaping their intended operating environments could negatively impact ecosystems and human health. To avoid breaches resulting from metabolic cross-feeding, horizontal gene transfer, and/or genetic mutations, synthetic auxotrophs have been engineered to become dependent on exogenously supplied xenobiotics, such as noncanonical amino acids (ncAAs). The incorporation of these abiological building blocks into essential proteins constitutes a first step toward constructing xenobiological barriers between GMOs and their environments. To transition synthetic auxotrophs further away from familiar biology, we demonstrate how bacterial growth can be confined by transition-metal complexes that catalyze the formation of an essential ncAA through new-to-nature reactions. Specifically, using a homogeneous ruthenium complex enabled us to localize bacterial growth on solid media, while heterogeneous palladium nanoparticles could be recycled and deployed up to five consecutive times to ensure the survival of synthetic auxotrophs in liquid cultures

Tethered Ribosomes:Toward the Synthesis of Nonproteinogenic Polymers in Bacteria

The ribosome is the core element of the translational apparatus and displays unrivaled fidelity and efficiency in the synthesis of long polymers with defined sequences and diverse compositions. Repurposing ribosomes for the assembly of nonproteinogenic (bio)polymers is an enticing prospect with implications for fundamental science, bioengineering and synthetic biology alike. Here, we review tethered ribosomes, which feature inseparable large and small subunits that can be evolved for novel function without interfering with native translation. Following a tutorial summary of ribosome structure, function, and biogenesis, we introduce design and optimization strategies for the creation of orthogonal and tethered ribosomes. We also highlight studies, in which (rational) engineering efforts of these designer ribosomes enabled the evolution of new functions. Lastly, we discuss future prospects and challenges that remain for the ribosomal synthesis of tailor-made (bio)polymers.</p

Genetic-Code-Expansion Strategies for Vaccine Development

Abstract: By providing long-term protection against infectious diseases, vaccinations have significantly reduced death and morbidity worldwide. In the 21st century, (bio)technological advances have paved the way for developing prophylactic vaccines that are safer and more effective as well as enabling the use of vaccines as therapeutics to treat human diseases. Here, we provide a focused review of the utility of genetic code expansion as an emerging tool for the development of vaccines. Specifically, we discuss how the incorporation of immunogenic noncanonical amino acids can aid in eliciting immune responses against adverse self-proteins and highlight the potential of an expanded genetic code for the construction of replication-incompetent viruses. We close the review by discussing the future prospects and remaining challenges for the application of these approaches in the development of both prophylactic and therapeutic vaccines in the near future

A Screening Platform to Identify and Tailor Biocompatible Small-Molecule Catalysts

Interfacing biocompatible, small-molecule catalysis with cellular metabolism promises a straightforward introduction of new function into organisms without the need for genetic manipulation. However, identifying and optimizing synthetic catalysts that perform new-to-nature transformations under conditions that support life is a cumbersome task. To enable the rapid discovery and fine-tuning of biocompatible catalysts, we describe a 96-well screening platform that couples the activity of synthetic catalysts to yield non-canonical amino acids from appropriate precursors with the subsequent incorporation of these nonstandard building blocks into GFP (quantifiable readout). Critically, this strategy does not only provide a common readout (fluorescence) for different reaction/catalyst combinations, but also informs on the organism's fitness, as stop codon suppression relies on all steps of the central dogma of molecular biology. To showcase our approach, we have applied it to the evaluation and optimization of transition-metal-catalyzed deprotection reactions

A Strategy to Select Macrocyclic Peptides Featuring Asymmetric Molecular Scaffolds as Cyclization Units by Phage Display

[Image: see text] Macrocyclic peptides (MPs) have positioned themselves as a privileged class of compounds for the discovery of therapeutics and development of chemical probes. Aided by the development of powerful selection strategies, high-affinity binders against biomolecular targets can readily be elicited from massive, genetically encoded libraries by affinity selection. For example, in phage display, MPs are accessed on the surface of whole bacteriophages via disulfide formation, the use of (symmetric) crosslinkers, or the incorporation of non-canonical amino acids. To facilitate a straightforward cyclization of linear precursors with asymmetric molecular scaffolds, which are often found at the core of naturally occurring MPs, we report an efficient two-step strategy to access MPs via the programmed modification of a unique cysteine residue and an N-terminal amine. We demonstrate that this approach yields MPs featuring asymmetric cyclization units from both synthetic peptides and when linear precursors are appended onto a phage-coat protein. Finally, we showcase that our cyclization strategy is compatible with traditional phage-display protocols and enables the selection of MP binders against a model target protein from naïve libraries. By enabling the incorporation of non-peptidic moieties that (1) can serve as cyclization units, (2) provide interactions for binding, and/or (3) tailor pharmacological properties, our head-to-side-chain cyclization strategy provides access to a currently under-explored chemical space for the development of chemical probes and therapeutics

An Epigenetics-Inspired DNA-Based Data Storage System.

Biopolymers are an attractive alternative to store and circulate information. DNA, for example, combines remarkable longevity with high data storage densities and has been demonstrated as a means for preserving digital information. Inspired by the dynamic, biological regulation of (epi)genetic information, we herein present how binary data can undergo controlled changes when encoded in synthetic DNA strands. By exploiting differential kinetics of hydrolytic deamination reactions of cytosine and its naturally occurring derivatives, we demonstrate how multiple layers of information can be stored in a single DNA template. Moreover, we show that controlled redox reactions allow for interconversion of these DNA-encoded layers of information. Overall, such interlacing of multiple messages on synthetic DNA libraries showcases the potential of chemical reactions to manipulate digital information on (bio)polymers.C.M. is grateful for the financial support by the Swiss National Science Foundation (grant number P2EZP2_152216). G.R.M. was supported by funding from Trinity College, Cambridge, the Herchel Smith fund and the Wellcome Trust. P.M. was funded by the Wellcome Trust and is currently supported by an ERC Advanced grant. P.V.D was funded by the Wellcome Trust and a Marie Curie Fellow of the European Union (grant number FP7-PEOPLE-2013-IEF/624885). The S.B. lab is supported by a program grant and core funding from Cancer Research UK (C9681/A18618), an ERC Advanced grant (339778) and by a Senior Investigator Award of the Wellcome Trust (099232/Z/12/Z). We thank Eun-Ang Raiber and Dario Beraldi for stimulating discussions and proofreading the manuscript.This is the final version of the article. It first appeared from Wiley at http://dx.doi.org/10.1002/anie.201605531

Affinity-Selected Bicyclic Peptide G-Quadruplex Ligands Mimic a Protein-like Binding Mechanism.

The study of G-quadruplexes (G4s) in a cellular context has demonstrated links between these nucleic acid secondary structures, gene expression, and DNA replication. Ligands that bind to the G4 structure therefore present an excellent opportunity for influencing gene expression through the targeting of a nucleic acid structure rather than sequence. Here, we explore cyclic peptides as an alternative class of G4 ligands. Specifically, we describe the development of de novo G4-binding bicyclic peptides selected by phage display. Selected bicyclic peptides display submicromolar affinity to G4 structures and high selectivity over double helix DNA. Molecular simulations of the bicyclic peptide-G4 complexes corroborate the experimental binding strengths and reveal molecular insights into G4 recognition by bicyclic peptides via the precise positioning of amino acid side chains, a binding mechanism reminiscent of endogenous G4-binding proteins. Overall, our results demonstrate that selection of (bi)cyclic peptides unlocks a valuable chemical space for targeting nucleic acid structures.Jesus College, Cambridge (Embiri- cos Scholarship) and the Herchel Smith studentship. The Balasubramanian group receives programme funding (C9681/A18618) and core funding (C14303/A17197) from Cancer Re- search UK, and an Investigator Award from the Wellcome Trust (099232/Z/12/Z). KR DJW received funding from the EPSRC (EP/N035003/1) and KR also received funding from the Cambridge Philosophical Societ

Quantifying the effect of ocean bed properties on ice sheet geometry over 40 000 years with a full-Stokes model

Simulations of ice sheet evolution over glacial cycles require integration of observational constraints using ensemble studies with fast ice sheet models. These include physical parameterisations with uncertainties, for example, relating to grounding-line migration. More complete ice dynamic models are slow and have thus far only be applied for  50 % under almost equal forcing. Grounding-line positions differ by up to 49 km, show significant hysteresis, and migrate non-steadily in both scenarios with long quiescent phases disrupted by leaps of rapid migration. The simulations quantify the evolution of two different ice sheet geometries (namely thick and slow vs. thin and fast), triggered by the variable grounding-line migration over the differing ocean beds. Our study extends the timescales of 3D full-Stokes by an order of magnitude compared to previous studies with the help of parallelisation. The extended time frame for full-Stokes models is a first step towards better understanding other processes such as erosion and sediment redistribution in the ice shelf cavity impacting the entire catchment geometry

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence‐related to other ATP‐dependent enzymes involved in lactam hydrolysis, like 5‐oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP‐dependent acetone‐ and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His‐tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6‐aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG(0)'= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)(2) tetramer and revealed an ATP‐binding site in CapA and a Zn‐coordinating site in CapB. Mutations in the ATP‐binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild‐type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP‐dependent lactam hydrolysis

Searching for TeV dark matter by atmospheric Cerenkov techniques

There is a growing interest in the possibility that dark matter could be formed of weakly interacting particles with a mass in the 100 GeV - 2 TeV range, and supersymmetric particles are favorite candidates. If they constitute the dark halo of our Galaxy, their mutual annihilations produce energetic gamma rays that could be detected using existing atmospheric \u{C}erenkov techniques.Comment: 10 pp, LaTex (3 figures available by e-mail) PAR-LPTHE 92X