Selective manipulation of superparamagnetic nanoparticles for product purification and microfluidic diagnostics.

The needs of scalable product purification as well as the demand for sensitive diagnostics for highly dilute entities can be addressed with the utilization of tailored superparamagnetic nanoparticles. Recent developments have led to more efficient fluidic systems at different scales with suspended nanoparticles or nanoparticle aggregates. However, magnetic nanoparticle systems differ widely in properties and their applications are characterized by very specific challenges.

Protein complex formation during denitrification by Pseudomonas aeruginosa.

The most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N O to molecular nitrogen (N ) by four membrane-localized reductases with the simultaneous formation of an ion gradient for ATP synthesis.

Biomacromolecules as tools and objects in nanometrology-current challenges and perspectives.

Nucleic acids, proteins, and polysaccharides are the most important classes of biopolymers. The inherent properties of biomacromolecules are contrary to those of well-defined small molecules consequently raising a number of specific challenges which become particularly apparent if biomacromolecules are treated as objects in quantitative analysis. At the same time, their specific functional ability of molecular recognition and self-organization (e.

Heme and nitric oxide binding by the transcriptional regulator DnrF from the marine bacterium increases promoter affinity.

Under oxygen-limiting conditions, the marine bacterium DFL12 generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF.

A DNA Walker as a Fluorescence Signal Amplifier.

Sensing nucleic acids typically involves the recognition of a specific sequence and reporting by, for example, a fluorogenic reaction yielding one activated dye molecule per detected nucleic acid. Here, we show that after binding to a DNA origami track a bound DNA target (a "DNA walker") can release the fluorescence of many molecules by acting as the catalyst of an enzymatic nicking reaction. As the walking kinetics sensitively depends on the walker sequence, the resulting brightness distribution of DNA origamis is a sequence fingerprint with single-nucleotide sensitivity.

BrEPS 2.0: Optimization of sequence pattern prediction for enzyme annotation.

The prediction of gene functions is crucial for a large number of different life science areas. Faster high throughput sequencing techniques generate more and larger datasets. The manual annotation by classical wet-lab experiments is not suitable for these large amounts of data.

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B.

Plasmonics Enhanced Smartphone Fluorescence Microscopy.

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses.

Broadband Fluorescence Enhancement with Self-Assembled Silver Nanoparticle Optical Antennas.

Plasmonic structures are known to affect the fluorescence properties of dyes placed in close proximity. This effect has been exploited in combination with single-molecule techniques for several applications in the field of biosensing. Among these plasmonic structures, top-down zero-mode waveguides stand out due to their broadband capabilities.

The BRENDA enzyme information system-From a database to an expert system.

Enzymes, representing the largest and by far most complex group of proteins, play an essential role in all processes of life, including metabolism, gene expression, cell division, the immune system, and others. Their function, also connected to most diseases or stress control makes them interesting targets for research and applications in biotechnology, medical treatments, or diagnosis. Their functional parameters and other properties are collected, integrated, and made available to the scientific community in the BRaunschweig ENzyme DAtabase (BRENDA).

630Δ and - quantitative growth and extensive polysaccharide secretion.

Antibiotic-associated infections with are a severe and often lethal risk for hospitalized patients, and can also affect populations without these classical risk factors. For a rational design of therapeutical concepts, a better knowledge of the metabolism of the pathogen is crucial. Metabolic modeling can provide a simulation of quantitative growth and usage of metabolic pathways, leading to a deeper understanding of the organism.

Manual curation and reannotation of the genomes of Clostridium difficile 630Δerm and C. difficile 630.

Purpose: We resequenced the genome of Clostridium difficile 630Δerm (DSM 28645), a model strain commonly used for the generation of insertion mutants.

Methodology: The genome sequence was obtained by a combination of single-molecule real-timeand Illumina sequencing technology.

Results: Detailed manual curation and comparison to the previously published genomic sequence revealed sequence differences including inverted regions and the presence of plasmid pCD630.

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process.

Shifting molecular localization by plasmonic coupling in a single-molecule mirage.

Over the last decade, two fields have dominated the attention of sub-diffraction photonics research: plasmonics and fluorescence nanoscopy. Nanoscopy based on single-molecule localization offers a practical way to explore plasmonic interactions with nanometre resolution. However, this seemingly straightforward technique may retrieve false positional information.

BRENDA in 2017: new perspectives and new tools in BRENDA.

The BRENDA enzyme database (www.brenda-enzymes.org) has developed into the main enzyme and enzyme-ligand information system in its 30 years of existence.

In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.

This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture.

Fluorescent Nanodiamond-Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging.

There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs.

A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains.

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle.

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive.

Complete genome sequence of the bioleaching bacterium Leptospirillum sp. group II strain CF-1.

We describe the complete genome sequence of Leptospirillum sp. group II strain CF-1, an acidophilic bioleaching bacterium isolated from an acid mine drainage (AMD). This work provides data to gain insights about adaptive response of Leptospirillum spp.

Superresolution microscopy with transient binding.

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels.

Multifunctional Dumbbell-Shaped DNA-Templated Selective Formation of Fluorescent Silver Nanoclusters or Copper Nanoparticles for Sensitive Detection of Biomolecules.

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine-thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation.

DNA Origami Nanoantennas with over 5000-fold Fluorescence Enhancement and Single-Molecule Detection at 25 μM.

Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration.

Super-Resolution Imaging Conditions for enhanced Yellow Fluorescent Protein (eYFP) Demonstrated on DNA Origami Nanorulers.

Photostability is one of the crucial properties of a fluorophore which strongly influences the quality of single molecule-based super-resolution imaging. Enhanced yellow fluorescent protein (eYFP) is one of the most widely used versions of fluorescent proteins in modern cell biology exhibiting fast intrinsic blinking and reversible photoactivation by UV light. Here, we developed an assay for studying photostabilization of single eYFP molecules with respect to fast blinking and demonstrated a 6-fold enhanced photostability of single eYFP molecules with a beneficial influence on the blinking kinetics under oxygen removal and addition of aliphatic thiols (dSTORM-buffer).

Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium.

The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium.