Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell-free gene expression systems. One of the crucial steps during the preparation of cell extract-based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell-free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108  PFU/mL. State-of-the-art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme-assisted sonication (LAS) extracts, but we observed an increase in the release of DNA-binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia.

BACKGROUND: Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. RESULTS: In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014-2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. CONCLUSIONS: K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.

Growth of Giant Peptide Vesicles Driven by Compartmentalized Transcription-Translation Activity.

Compartmentalization and spatial organization of biochemical reactions are essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosures of artificial cell mimics. Herein, we report on the formation of giant vesicles in a size range of 1 µm-100 µm using amphiphilic elastin-like polypeptides. The peptide vesicles can accommodate cell-free gene expression reactions, which is demonstrated by the transcription of a fluorescent RNA aptamer and the production of a fluorescent protein. Importantly, gene expression inside the vesicles leads to a strong growth of their size-up to an order of magnitude in volume in several cases-which is driven by changes in osmotic pressure, resulting in fusion events and uptake of membrane peptides from the environment.

Periodic Operation of a Dynamic DNA Origami Structure Utilizing the Hydrophilic-Hydrophobic Phase-Transition of Stimulus-Sensitive Polypeptides.

Dynamic DNA nanodevices are designed to perform structure-encoded motion actuated by a variety of different physicochemical stimuli. In this context, hybrid devices utilizing other components than DNA have the potential to considerably expand the library of functionalities. Here, the reversible reconfiguration of a DNA origami structure using the stimulus sensitivity of elastin-like polypeptides is reported. To this end, a rectangular sheet made using the DNA origami technique is functionalized with these peptides and by applying changes in salt concentration the hydrophilic-hydrophobic phase transition of these peptides actuate the folding of the structure. The on-demand and reversible switching of the rectangle is driven by externally imposed temperature oscillations and appears at specific transition temperatures. Using transmission electron microscopy, it is shown that the structure exhibits distinct conformational states with different occupation probabilities, which are dependent on structure-intrinsic parameters such as the local number and the arrangement of the peptides on the rectangle. It is also shown through ensemble fluorescence resonance energy transfer spectroscopy that the transition temperature and thus the thermodynamics of the rectangle-peptide system depends on the stimuli salt concentration and temperature, as well as on the intrinsic parameters.

Enhanced Efficiency of an Enzyme Cascade on DNA-Activated Silica Surfaces.

In nature, compartmentalized and spatially organized enzyme cascades are utilized to increase the efficiency of enzymatic reactions. From a technologically relevant perspective, synthetic enzyme systems have to be optimized with emphasis on enzyme activity, productivity, scalability, and ease of use. But the underlying principles and relevant parameters that lead to an enhancement of the activity of enzyme cascades through spatial organization are still under debate. Here, we report on the 10-fold activity enhancement of the GOx-HRP enzyme cascade for the oxidation of luminol, when the enzymes are colocalized on micron-scaled solid scaffolds. Both enzymes were initially assembled and concentrated on DNA origami rectangles and finally further concentrated on the surface of silica particles. We show that each particular component of the designed system contributes to the activity enhancement. Furthermore, we measured an influence of the silica particle length scale on the total productivity by a factor of 5-10, but to a lesser extent on the maximum enzyme activity. Our findings demonstrate that micrometer-sized scaffolds can be used to enhance the efficiency of enzyme-cascades by at least a magnitude and that solid-phase scaffolds enable scalability for technological applications.

Formation of vesicular structures from fatty acids formed under simulated volcanic hydrothermal conditions.

Microscopic compartmentalization is beneficial in synthetic chemistry and indispensable for the evolution of life to separate a reactive "inside" from a hydrolyzing "outside". Here, we show compartmentalization in aqueous solution containing mixtures of fatty acids up to 19 carbon atoms which were synthesized by one-pot reactions of acetylene and carbon monoxide in contact with nickel sulfide at 105 °C, reaction requirements which are compatible to Hadean Early Earth conditions. Based on confocal, dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, vesicle-like structures with diameters of 10-150 nm are formed after solvent extraction and resolubilisation. Moreover fluorescent dye was encapsulated into the structures proving their vesicular properties. This self-assembly could also have occurred on Early Earth as a crucial step in establishing simple membranes of proto-cells as a prerequisite in the evolution of metabolism and life.

Phage Therapy in Germany-Update 2023.

Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages.

Practical Assessment of an Interdisciplinary Bacteriophage Delivery Pipeline for Personalized Therapy of Gram-Negative Bacterial Infections.

Despite numerous advances in personalized phage therapy, smooth logistics are challenging, particularly for multidrug-resistant Gram-negative bacterial infections requiring high numbers of specific lytic phages. We conducted this study to pave the way for efficient logistics for critically ill patients by (1) closely examining and improving a current pipeline under realistic conditions, (2) offering guidelines for each step, leading to safe and high-quality phage supplies, and (3) providing a tool to evaluate the pipeline's efficiency. Due to varying stipulations for quality and safety in different countries, we focused the pipeline on all steps up to a required phage product by a cell-free extract system. The first of three study runs included patients with respiratory bacterial infections from four intensive care units, and it revealed a cumulative time of up to 23 days. Ultimately, adjustment of specific set points of the vulnerable components of the pipeline, phage isolation, and titration increased the pipeline's efficiency by 15% and decreased the maximum required time to 13 days. We present a site-independent practical approach to establish and optimize pipelines for personalized phage delivery, the co-organization of pipeline components between different institutions, non-binding guidelines for every step, and an efficiency check for phage laboratories.

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

Small Antisense DNA-Based Gene Silencing Enables Cell-Free Bacteriophage Manipulation and Genome Replication.

Cell-free systems allow interference with gene expression processes without requiring elaborate genetic engineering procedures. This makes it ideally suited for rapid prototyping of synthetic biological parts. Inspired by nature's strategies for the control of gene expression via short antisense RNA molecules, we here investigated the use of small DNA (sDNA) for translational inhibition in the context of cell-free protein expression. We designed sDNA molecules to be complementary to the ribosome binding site (RBS) and the downstream coding sequence of targeted mRNA molecules. Depending on sDNA concentration and the promoter used for transcription of the mRNA, this resulted in a reduction of gene expression of targeted genes by up to 50-fold. We applied the cell-free sDNA technique (CF-sDNA) to modulate cell-free gene expression from the native T7 phage genome by suppressing the production of the major capsid protein of the phage. This resulted in a reduced phage titer, but at the same time drastically improved cell-free replication of the phage genome, which we utilized to amplify the T7 genome by more than 15 000-fold in a droplet-based serial dilution experiment. Our simple antisense sDNA approach extends the possibilities to exert translational control in cell-free expression systems, which should prove useful for cell-free prototyping of native phage genomes and also cell-free phage manipulation.

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Towards synthetic cells using peptide-based reaction compartments.

Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell's genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.

Self-Assembled Active Plasmonic Waveguide with a Peptide-Based Thermomechanical Switch.

Nanoscale plasmonic waveguides composed of metallic nanoparticles are capable of guiding electromagnetic energy below the optical diffraction limit. Signal feed-in and readout typically require the utilization of electronic effects or near-field optical techniques, whereas for their fabrication mainly lithographic methods are employed. Here we developed a switchable plasmonic waveguide assembled from gold nanoparticles (AuNPs) on a DNA origami structure that facilitates a simple spectroscopic excitation and readout. The waveguide is specifically excited at one end by a fluorescent dye, and energy transfer is detected at the other end via the fluorescence of a second dye. The transfer distance is beyond the multicolor FRET range and below the Abbé limit. The transmittance of the waveguide can also be reversibly switched by changing the position of a AuNP within the waveguide, which is tethered to the origami platform by a thermoresponsive peptide. High-yield fabrication of the plasmonic waveguides in bulk was achieved using silica particles as solid supports. Our findings enable bulk solution applications for plasmonic waveguides as light-focusing and light-polarizing elements below the diffraction limit.