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1.
Nat Biotechnol ; 42(2): 175-178, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38361070
6.
Commun Biol ; 5(1): 675, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35798943

RESUMO

Although the essential proteins that drive bacterial cytokinesis have been identified, the precise mechanisms by which they dynamically interact to enable symmetrical division are largely unknown. In Escherichia coli, cell division begins with the formation of a proto-ring composed of FtsZ and its membrane-tethering proteins FtsA and ZipA. In the broadly proposed molecular scenario for ring positioning, Min waves composed of MinD and MinE distribute the FtsZ-polymerization inhibitor MinC away from mid-cell, where the Z-ring can form. Therefore, MinC is believed to be an essential element connecting the Min and FtsZ subsystems. Here, by combining cell-free protein synthesis with planar lipid membranes and microdroplets, we demonstrate that MinDE drive the formation of dynamic, antiphase patterns of FtsA-anchored FtsZ filaments even in the absence of MinC. These results suggest that Z-ring positioning may be achieved with a more minimal set of proteins than previously envisaged, providing a fresh perspective about synthetic cell division.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Proteínas de Escherichia coli , Proteínas de Membrana , Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo
7.
ACS Synth Biol ; 10(10): 2447-2455, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34585918

RESUMO

Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB, which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by a light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.


Assuntos
Lipossomos , Microtúbulos/metabolismo , Verrucomicrobia/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Livre de Células , Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo
8.
Sci Rep ; 11(1): 1898, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479285

RESUMO

The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 × 10-3 and 13.2 × 10-3, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.


Assuntos
DNA/genética , Biossíntese de Proteínas/genética , RNA/genética , Proteínas Recombinantes/biossíntese , Aminoácidos/genética , Aminoacil-tRNA Sintetases , Sistema Livre de Células , Códon/genética , Escherichia coli/genética , Fases de Leitura Aberta , Fator Tu de Elongação de Peptídeos/genética , Proteômica/métodos , Proteínas Recombinantes/genética , Ribossomos/genética , Transcrição Gênica/genética
9.
Commun Biol ; 3(1): 539, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999429

RESUMO

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.


Assuntos
Células Artificiais/metabolismo , Divisão Celular , Escherichia coli/fisiologia , Lipossomos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Sistema Livre de Células/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo
10.
Nat Commun ; 10(1): 4969, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672986

RESUMO

The Min biochemical network regulates bacterial cell division and is a prototypical example of self-organizing molecular systems. Cell-free assays relying on purified proteins have shown that MinE and MinD self-organize into surface waves and oscillatory patterns. In the context of developing a synthetic cell from elementary biological modules, harnessing Min oscillations might allow us to implement higher-order cellular functions. To convey hereditary information, the Min system must be encoded in a DNA molecule that can be copied, transcribed, and translated. Here, the MinD and MinE proteins are synthesized de novo from their genes inside liposomes. Dynamic protein patterns and accompanying liposome shape deformation are observed. When integrated with the cytoskeletal proteins FtsA and FtsZ, the synthetic Min system is able to dynamically regulate FtsZ patterns. By enabling genetic control over Min protein self-organization and membrane remodeling, our methodology offers unique opportunities towards directed evolution of bacterial division processes in vitro.


Assuntos
Adenosina Trifosfatases/metabolismo , Células Artificiais/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Ácidos Nucleicos Livres , Escherichia coli , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas , Lipossomos/ultraestrutura , Proteínas de Membrana/genética
11.
Phys Biol ; 16(2): 025001, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30625117

RESUMO

DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (protein synthesis using recombinant elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, there is an urgent need to improve the system's performance and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors (IFs) by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, the depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNA-hydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.


Assuntos
Ácidos Nucleicos Livres/biossíntese , Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , RNA Bacteriano/biossíntese , Modelos Químicos
12.
Sci Rep ; 7(1): 16094, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170466

RESUMO

The inherent stochasticity of molecular reactions prevents us from predicting the exact state of single-cells in a population. However, when a population grows at steady-state, the probability to observe a cell with particular combinations of properties is fixed. Here we validate and exploit existing theory on the statistics of single-cell growth in order to predict the probability of phenotypic characteristics such as cell-cycle times, volumes, accuracy of division and cell-age distributions, using real-time imaging data for Bacillus subtilis and Escherichia coli. Our results show that single-cell growth-statistics can accurately be predicted from a few basic measurements. These equations relate different phenotypic characteristics, and can therefore be used in consistency tests of experimental single-cell growth data and prediction of single-cell statistics. We also exploit these statistical relations in the development of a fast stochastic-simulation algorithm of single-cell growth and protein expression. This algorithm greatly reduces computational burden, by recovering the statistics of growing cell-populations from the simulation of only one of its lineages. Our approach is validated by comparison of simulations and experimental data. This work illustrates a methodology for the prediction, analysis and tests of consistency of single-cell growth and protein expression data from a few basic statistical principles.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Algoritmos , Bacillus subtilis/citologia , Escherichia coli/citologia , Modelos Teóricos
13.
Biochim Biophys Acta Mol Cell Res ; 1864(1): 231-242, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27818273

RESUMO

Non-dividing Saccharomyces cerevisiae cultures are highly relevant for fundamental and applied studies. However, cultivation conditions in which non-dividing cells retain substantial metabolic activity are lacking. Unlike stationary-phase (SP) batch cultures, the current experimental paradigm for non-dividing yeast cultures, cultivation under extreme calorie restriction (ECR) in retentostat enables non-dividing yeast cells to retain substantial metabolic activity and to prevent rapid cellular deterioration. Distribution of F-actin structures and single-cell copy numbers of specific transcripts revealed that cultivation under ECR yields highly homogeneous cultures, in contrast to SP cultures that differentiate into quiescent and non-quiescent subpopulations. Combined with previous physiological studies, these results indicate that yeast cells subjected to ECR survive in an extended G1 phase. This study demonstrates that yeast cells exposed to ECR differ from carbon-starved cells and offer a promising experimental model for studying non-dividing, metabolically active, and robust eukaryotic cells.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Metabolismo Energético/genética , Regulação Fúngica da Expressão Gênica , Glucose/deficiência , Saccharomyces cerevisiae/metabolismo , Actinas/genética , Actinas/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Meios de Cultura/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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