Biology subjects

Niederholtmeyer, H.

Publications and source records attributed to Niederholtmeyer, H..

8 recordsLinked to original sources

bioRxiv2026

Tardigrade-Derived Strategy for Low-Cost Storage of Cell-Free Expression Lysates

Cell-free expression systems (CFES) are increasingly used alongside conventional biotechnological approaches to accelerate early-stage prototyping and are particularly valuable in point-of-use settings. However, their broader adoption remains limited by time- and cost-intensive preparation, as well as stringent cryogenic storage requirements. To address this, several studies have explored lyophilization with protective additives to generate stable, solid-state CFES. These approaches had to balance the protection gained with a loss of activity due to the additives. In this study, we present a CFES that contains a tardigrade-derived Cytosolic-Abundant Heat-Soluble (CAHS) protein to protect the biosynthetic machinery in lysates from damages during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity during low-cost room temperature desiccation, while unprotected lysates are affected in mRNA synthesis kinetics and translation yields. The diversity of tardigrade-derived protective proteins is a treasure trove for cell-free synthetic biology, in particular for making CFES more accessible and portable. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/715078v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11cd5b7org.highwire.dtl.DTLVardef@130c02org.highwire.dtl.DTLVardef@795c78org.highwire.dtl.DTLVardef@1a5ffdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

RNA-based communication in heterogeneous populations of cell mimics

RNA regulators offer a promising path for building complex, orthogonal circuits due to their low resource demands and design flexibility. In this study we explore their potential as signaling molecules in communication between synthetic cells. Specifically, we engineer populations of heterogenetic porous polymer cell mimics to produce, emit and receive two types of small synthetic RNA regulators. These RNAs are required to activate reporter expression at both the levels of transcription and translation. We distribute this AND gate circuit in receiver and two types of sender cell mimics to compare the distributed logic computation to the behavior of the circuit in well-mixed, bulk cell-free expression reactions. Analyzing different densities and spatial arrangements of senders and receivers, we reveal spatiotemporal gradients in RNA signals and identify configurations that increase specific activation. With small regulatory RNAs, the engineering toolbox for communication between synthetic cells expands to include a programmable class of signaling molecules. The rapid turnover of RNA suggests applications in establishing dynamic signaling gradients in communities of synthetic cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/673985v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@3892b3org.highwire.dtl.DTLVardef@2751feorg.highwire.dtl.DTLVardef@1780922org.highwire.dtl.DTLVardef@19fc511_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy timescales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase, as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5 and 3 untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5UTRs, 10 3UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/580994v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a6b24org.highwire.dtl.DTLVardef@2b62org.highwire.dtl.DTLVardef@1203308org.highwire.dtl.DTLVardef@f674a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

A cell-free system for target discovery of small membrane proteins

Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production challenging, hindering function discovery. Here, we combined a cell-free system with lipid sponge droplets and synthesized small membrane proteins in vitro. Lipid sponge droplets contain a dense network of lipid bilayers, which accommodates and extracts newly synthesized small membrane proteins from the aqueous surroundings. Using small bacterial membrane proteins MgrB, SafA, and AcrZ as proof of principle, we showed that the in vitro produced membrane proteins were functionally active, for example, modulating the activity of their target kinase as expected. The cell-free system produced small membrane proteins, including one from human, up to micromolar concentrations, indicating its high level of versatility and productivity. Furthermore, AcrZ produced in this system was used successfully for in vitro co-immunoprecipitations to identify interaction partners. This work presents a robust alternative approach for producing small membrane proteins, which opens a door to their function discovery in different domains of life. ImportanceSmall membrane proteins are shown to be involved in various biological processes in all domains of life and "can no longer be ignored". Due to their small size and hydrophobicity, functional investigation of small membrane proteins is challenging. In this work, we present a simple, versatile, cell-free approach for synthesizing small membrane proteins in vitro. We show that the small membrane proteins produced with our system are functional and in sufficient amounts for downstream target discoveries. Furthermore, our approach may uncover additional regulatory functions of small membrane proteins studied with conventional methods. Our work provides a robust alternative workflow for functional studies, which opens up new possibilities to advance our understanding of small membrane protein biology.

synthetic biology↗

bioRxiv2022

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions. Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown. Here, we characterize the cell-free production of infectious bacteriophage T7 virions under simulated microgravity. Rotation in a 2D-clinostat increased the number of infectious particles compared to static controls. Quantitative analyses by mass spectrometry, immuno-dot-blot and real-time PCR showed no significant differences in protein and DNA contents, suggesting enhanced self-assembly of T7 phages in simulated microgravity. While the effects of genuine space conditions on the cell-free synthesis and assembly of bacteriophages remain to be investigated, our findings support the vision of a cell-free synthesis-enabled "astropharmacy".

synthetic biology↗

bioRxiv2021

Functionalizing lipid sponge droplets with DNA

Nucleic acids are among the most versatile molecules for the construction of biomimetic systems because they can serve as information carriers and programmable construction materials. How nucleic acids interact with membranous coacervate compartments such as lipid sponge droplets is not known. Here we systematically characterize the potential of DNA to functionalize lipid sponge droplets and demonstrate a strong size dependence for sequestration into the sponge phase. Double stranded DNA molecules of more than 300 bp are excluded and form a corona on the surface of droplets they are targeted to. Shorter DNA molecules partition efficiently into the lipid sponge phase and can direct DNA-templated reactions to droplets. We demonstrate repeated capture and release of labeled DNA strands by dynamic hybridization and strand displacement reactions that occur inside droplets. Our system opens new opportunities for DNA-encoded functions in lipid sponge droplets such as cargo control and signaling.

synthetic biology↗

bioRxiv2020

Lipid Sponge Droplets as Programmable Synthetic Organelles

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and non-ionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems. Significance statementOrganelles spatially and temporally orchestrate biochemical reactions in a cell to a degree of precision that is still unattainable in synthetic reaction systems. Additionally, organelles such as the endoplasmic reticulum (ER) contain highly interconnected and dense membrane networks that provide large reaction spaces for both transmembrane and soluble enzymes. We present lipid sponge droplets to emulate the functions of organelles such as the ER. We demonstrate that lipid sponge droplets can be programmed to internally concentrate specific proteins, host and accelerate biochemical transformations, and to rapidly and reversibly sequester and release proteins to control enzymatic reactions. The self-assembled and programmable nature of lipid sponge droplets will facilitate the integration of complex functions for bottom up synthetic biology.

synthetic biology↗

bioRxiv2018

Communication and quorum sensing in non-living mimics of eukaryotic cells

Cells in tissues or biofilms communicate with one another through chemical and mechanical signals to coordinate collective behaviors. Non-living cell mimics provide simplified models of natural systems, however, it has remained challenging to implement communication capabilities comparable to living cells. Here we present a porous artificial cell-mimic containing a nucleus-like DNA-hydrogel compartment that is able to express and display proteins, and communicate with neighboring cell-mimics through diffusive protein signals. We show that communication between cell-mimics allows distribution of tasks, quorum sensing, and cellular differentiation according to local environment. Cell-mimics can be manufactured in large quantities, easily stored, chemically modified, and spatially organized into diffusively connected tissue-like arrangements, offering a means for studying communication in large ensembles of artificial cells.

synthetic biology↗