RESUMO
In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, k(ss) cat, â¼ 28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.
Assuntos
Aptâmeros de Nucleotídeos/genética , Evolução Molecular Direcionada , RNA Catalítico/genética , DNA/genética , Microfluídica , RNA Catalítico/isolamento & purificação , Técnica de Seleção de AptâmerosRESUMO
We present a high-throughput droplet-based microfluidic analysis/screening platform for directed evolution of CotA laccase: droplet-based microfluidic modules were combined to develop an efficient system that allows cell detection and sorting based on the enzymatic activity. This platform was run on two different operating modes: the "analysis" mode allowing the analysis of the enzymatic activity in droplets at very high rates (>1000 Hz) and the "screening" mode allowing sorting of active droplets at 400 Hz. The screening mode was validated for the directed evolution of the cytoplasmic CotA laccase from B. subtilis, a potential interesting thermophilic cathodic catalyst for biofuel cells. Single E. coli cells expressing either the active CotA laccase (E. coli CotA) or an inactive frameshifted variant (E. coli ΔCotA) were compartmentalized in aqueous droplets containing expression medium. After cell growth and protein expression within the droplets, a fluorogenic substrate was "picoinjected" in each droplet. Fluorescence-activated droplet sorting was then used to sort the droplets containing the desired activity and the corresponding cells were then recultivated and identified using colorimetric assays. We demonstrated that E. coli CotA cells were enriched 191-fold from a 1 : 9 initial ratio of E. coli CotA to E. coli ΔCotA cells (or 437-fold from a 1 : 99 initial ratio) using a sorting rate of 400 droplets per s. This system allows screening of 10(6) cells in only 4 h, compared to 11 days for screening using microtitre plate-based systems. Besides this low error rate sorting mode, the system can also be used at higher throughputs in "enrichment" screening mode to make an initial purification of a library before further steps of selection. Analysis mode, without sorting, was used to rapidly quantify the activity of a CotA library constructed using error-prone PCR. This mode allows analysis of 10(6) cells in only 1.5 h.
Assuntos
Bacillus subtilis/enzimologia , Evolução Molecular Direcionada/instrumentação , Escherichia coli/enzimologia , Lacase/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Micromanipulação/instrumentação , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Desenho de Equipamento , Escherichia coli/genética , Escherichia coli/metabolismo , Citometria de Fluxo/instrumentação , Expressão Gênica , Ensaios de Triagem em Larga Escala/instrumentação , Lacase/genética , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Chronic osteomyelitis is a disease process that is characterized not infrequently by periods of clinical quiescence interspersed by symptomatic episodes of varying duration and severity. These periods of clinical quiescence have been attributed to several possible factors, including effective host defenses that keep the process at bay as well as glycocalyceal sequestration of the implicated pathogen. Recent work has demonstrated a potential third explanation for this phenomenon, that is, intracellular incorporation of the pathogen within the host osteoblast. This is a report of a successful osteoblast cell culture demonstrating the facultative intraosteoblastic location of a human osteomyelitis Staphylococcus aureus isolate as well as its microscopic features.
Assuntos
Osteoblastos/microbiologia , Osteomielite/microbiologia , Staphylococcus aureus/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Osteoblastos/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, â¼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.
Assuntos
Relógios Circadianos/genética , Metaboloma/genética , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Células Cultivadas , Ritmo Circadiano/genética , Creatina/metabolismo , Criptocromos/metabolismo , Redes Reguladoras de Genes , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Nitrogênio/metabolismo , Fatores de TempoRESUMO
The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.
Assuntos
Células Artificiais/metabolismo , Origem da Vida , RNA/biossíntese , Biocatálise , Endorribonucleases/química , Gotículas Lipídicas/química , Modelos Estatísticos , Conformação de Ácido Nucleico , Q beta Replicase/química , RNA/química , RNA Catalítico/química , Processos EstocásticosRESUMO
Staphylococcus aureus capsular polysaccharides are believed to play a role in adhesion to surfaces and may contribute to their antimicrobial resistance, thereby increasing the rates and severity of associated infections. The purpose of this study was to compare the adhesiveness of distinct S. aureus capsular polysaccharides to determine whether adhesiveness was a general or specific feature across different S. aureus strains. Atomic force microscopy was used to confirm the presence or absence of capsular polysaccharides and to measure adhesive forces on a noncapsulated, serotype 8, and serotype 2 strain of S. aureus. Serotype 8 displayed a larger range of adhesive forces (1-19 nN) than the noncapsulated (0-4 nN) and serotype 2 (0-4 nN) strain. The majority of adhesive forces for serotype 8 were in the 10-15 nN range. Removal of capsular polysaccharides gave a marked decrease in adhesive forces measured for serotype 8 and, to a lesser extent, a decrease for serotype 2. Noncapsulated, serotype 8, and serotype 2 S. aureus had water contact angles of 23.8 (+/-8.9), 34.4 (+/-2.5), and 56.7 (+/-11.2) degrees (mean +/- standard deviation), respectively. For the first time, capsular polysaccharides from serotype 8 (clinically common) and serotype 2 (clinically rare) were demonstrated to have different physical properties, which may account for variations in studies in which clinical isolates are utilized, and the conflict in proposed roles for capsular polysaccharides on S. aureus is explained.
Assuntos
Aderência Bacteriana , Cápsulas Bacterianas/ultraestrutura , Microscopia de Força Atômica , Staphylococcus aureus/ultraestrutura , Fenômenos Biomecânicos , Modelos Biológicos , Propriedades de Superfície , ÁguaRESUMO
The mechanical response, the force-indentation relationship, in normal force spectroscopy measurements carried out on individual polysaccharide encapsulated bacteria is modeled using three increasingly refined approaches that consider the elastic response of the bacterium and cantilever in combination with a fluid (hydrodynamic) model for the polysaccharide layer. For the hydrodynamic description of the polysaccharide layer, several increasingly realistic models are described in detail, together with numerical solution techniques. These models range from one-dimensional, Newtonian, to two-dimensional, axisymmetric, fully viscoelastic (Phan-Thien/Tanner). In all cases, the models rigorously consider the time-dependent rheological-mechanical coupling between the elastic and fluid viscoelastic physical components of the experimental setup. Effects of inherent variability in geometrical and material properties of the bacterium and polysaccharide layer on the measurable response are quantified. A parametric investigation of the force-indentation relationship highlights the importance of accurate knowledge of the rheology of the extracellular polysaccharides. We also draw conclusions about the design and evaluation of force spectroscopy experiments on single encapsulated bacteria. Supported by model calculations, we also point the way to methods of in vivo rheological characterization of the extracellular polysaccharide as a preferable alternative to characterization after its removal from the native environment.
Assuntos
Polímeros/química , Análise Espectral/métodos , Bactérias/metabolismo , Biofísica/métodos , Físico-Química/métodos , Desenho de Equipamento , Mecânica , Microscopia de Força Atômica/métodos , Modelos Estatísticos , Modelos Teóricos , Polissacarídeos/química , Reologia , Propriedades de Superfície , Fatores de Tempo , ViscosidadeRESUMO
In this paper, experimentally obtained force curves on Staphylococcus aureus are compared with a previously developed model that incorporates hydrodynamic effects of extracellular polysaccharides together with the elastic response of the bacterium and cantilever. Force-displacement curves were predicted without any adjustable parameters. It is demonstrated that experimental results can be accurately described by our model, especially if viscoelastic effects of the extracellular polysaccharide layer are taken into account. Polysaccharide layer viscoelasticity was treated by means of a multimode Phan-Thien/Tanner (PTT) constitutive equation. Typical maximum relaxation times range from 0.2 to 2 s, whereas the corresponding zero-shear-rate viscosities are 6-9 Pa.s, based on published, steady-state rheological measurements on Staphylococcus aureus polysaccharide extracted from its native environment. The bacterial elastic constant is found to be in the range 0.02-0.4 N/m, corresponding to bacterial wall Young's moduli in the range of a few hundred MPa. Repeatability of measurements performed on different bacteria is found to be only fair, due to large individuum variability, whereas repetitions of measurements on the same bacterium showed high reproducibility. Improved force-indentation curve predictions are expected if transient rheological characterization of extracellular polysaccharides is available. More desirable however is the direct, in vivo rheological characterization of the extracellular polysaccharide. A model-based analysis of experimental force-indentation curves shows that appreciable further experimental improvements are still necessary to achieve this goal.