Light-Controlled Secretion and Injection of Proteins into Eukaryotic Cells by Optogenetic Control of the Bacterial Type III Secretion System.

Gram-negative bacteria can use the type III secretion system (T3SS) to inject effector proteins into eukaryotic target cells. In this chapter, we describe the application of a light-controlled T3SS, based on the targeted sequestration of an essential dynamic T3SS component with the help of optogenetic interaction switches. This method enables to control the secretion or injection into eukaryotic cells for a wide range of protein cargos with high temporal and spatial precision.

Impact of absolute values and changes in meteorological and air quality conditions on community-acquired pneumonia in Germany.

Community-acquired pneumonia (CAP) is a major global health concern as it is a leading cause of morbidity, mortality and economic burden to the health care systems. In Germany, more than 15,000 people die every year from CAP. Climate change is altering weather patterns, and it may influence the probability and severity of CAP.

Development of mirror-image monobodies targeting the oncogenic BCR::ABL1 kinase.

Mirror-image proteins, composed of D-amino acids, are an attractive therapeutic modality, as they exhibit high metabolic stability and lack immunogenicity. Development of mirror-image binding proteins is achieved through chemical synthesis of D-target proteins, phage display library selection of L-binders and chemical synthesis of (mirror-image) D-binders that consequently bind the physiological L-targets. Monobodies are well-established synthetic (L-)binding proteins and their small size (~90 residues) and lack of endogenous cysteine residues make them particularly accessible to chemical synthesis.

The Dual Roles of Lamin A/C in Macrophage Mechanotransduction.

Cellular mechanotransduction is a complex physiological process that integrates alterations in the external environment with cellular behaviours. In recent years, the role of the nucleus in mechanotransduction has gathered increased attention. Our research investigated the involvement of lamin A/C, a component of the nuclear envelope, in the mechanotransduction of macrophages under compressive force.

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme.

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs.

New-to-nature CO-dependent acetyl-CoA assimilation enabled by an engineered B-dependent acyl-CoA mutase.

Acetyl-CoA is a key metabolic intermediate and the product of various natural and synthetic one-carbon (C1) assimilation pathways. While an efficient conversion of acetyl-CoA into other central metabolites, such as pyruvate, is imperative for high biomass yields, available aerobic pathways typically release previously fixed carbon in the form of CO. To overcome this loss of carbon, we develop a new-to-nature pathway, the Lcm module, in this study.

Layered entrenchment maintains essentiality in the evolution of Form I Rubisco complexes.

Protein complexes composed of strictly essential subunits are abundant in nature and often arise through the gradual complexification of ancestral precursor proteins. Essentiality can arise through the accumulation of changes that are tolerated in the complex state but would be deleterious for the standalone complex components. While this theoretical framework to explain how essentiality arises has been proposed long ago, it is unclear which factors cause essentiality to persist over evolutionary timescales.

Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference.

CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase.

The mitochondrial citrate synthase from Tetrahymena thermophila does not form an intermediate filament.

The mitochondrial citrate synthase (mCS) purified from the ciliate Tetrahymena thermophila has been reported to form intermediate-filament-like structures during conjugation and to self-assemble into fibers when recombinantly expressed. This would represent a rare example of a tractable and recent origin of a novel cytoskeletal element. In an attempt to investigate the evolutionary emergence of this behavior, we re-investigated the ability of Tetrahymena's mCS to form filaments in vivo.

transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade.

metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing, and metabolic engineering. Yet, the analysis of the activity of such networks is very often restricted by time- and cost-intensive methods. To overcome these limitations, we sought to develop an transcription (IVT)-based biosensing workflow that is compatible with the complex conditions of metabolism, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a 27-component metabolic system that converts CO into glycolate.

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication.

Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences.

Mitochondria, Peroxisomes and Beyond-How Dual Targeting Regulates Organelle Tethering.

Eukaryotic cells feature distinct membrane-enclosed organelles such as mitochondria and peroxisomes, each playing vital roles in cellular function and organization. These organelles are linked at membrane contact sites, facilitating interorganellar molecule and ion exchange. Most contact-forming proteins identified to date are membrane proteins or membrane-associated proteins, which can form very stable contacts.

Golden Gate Cloning for the Standardized Assembly of Gene Elements with Modular Cloning in Chlamydomonas.

Modern synthetic biology requires fast and efficient cloning strategies for the assembly of new transcription units or entire pathways. Modular Cloning (MoClo) is a standardized synthetic biology workflow, which has tremendously simplified the assembly of genetic elements for transgene expression. MoClo is based on Golden Gate Assembly and allows to combine genetic elements of a library through a hierarchical syntax-driven pipeline.

Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.

Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool.

Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.

Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis.

Golden Gate Cloning of Synthetic CRISPR RNA Spacer Sequences.

Prokaryotes use CRISPR-Cas systems to interfere with viruses and other mobile genetic elements. CRISPR arrays comprise repeated DNA elements and spacer sequences that can be engineered for custom target sites. These arrays are transcribed into precursor CRISPR RNAs (pre-crRNAs) that undergo maturation steps to form individual CRISPR RNAs (crRNAs).

Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning.

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon.

Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox.

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand.

Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly.

Golden Gate cloning has become a powerful and widely used DNA assembly method. Its modular nature and the reusability of standardized parts allow rapid construction of transcription units and multi-gene constructs. Importantly, its modular structure makes it compatible with laboratory automation, allowing for systematic and highly complex DNA assembly.

Daily life in the Open Biologist's second job, as a Data Curator.

Background: Data reusability is the driving force of the research data life cycle. However, implementing strategies to generate reusable data from the data creation to the sharing stages is still a significant challenge. Even when datasets supporting a study are publicly shared, the outputs are often incomplete and/or not reusable.

Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis.