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1.
Science ; 385(6705): 174-178, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38991083

RESUMO

One of the hallmarks of living organisms is their capacity for self-organization and regeneration, which requires a tight integration of metabolic and genetic networks. We sought to construct a linked metabolic and genetic network in vitro that shows such lifelike behavior outside of a cellular context and generates its own building blocks from nonliving matter. We integrated the metabolism of the crotonyl-CoA/ethyl-malonyl-CoA/hydroxybutyryl-CoA cycle with cell-free protein synthesis using recombinant elements. Our network produces the amino acid glycine from CO2 and incorporates it into target proteins following DNA-encoded instructions. By orchestrating ~50 enzymes we established a basic cell-free operating system in which genetically encoded inputs into a metabolic network are programmed to activate feedback loops allowing for self-integration and (partial) self-regeneration of the complete system.


Assuntos
Sistema Livre de Células , Redes e Vias Metabólicas , Biossíntese de Proteínas , Glicina/metabolismo , Redes Reguladoras de Genes , Escherichia coli/metabolismo , Escherichia coli/genética , Dióxido de Carbono/metabolismo , Acil Coenzima A/metabolismo , Retroalimentação Fisiológica
2.
Nat Protoc ; 17(12): 2920-2965, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36261631

RESUMO

Droplet microfluidics is a powerful tool for a variety of biological applications including single-cell genetics, antibody discovery and directed evolution. All these applications make use of genetic libraries, illustrating the difficulty of generating chemically distinct droplets for screening applications. This protocol describes our Braille Display valving platform for on-demand generation of droplets with different chemical contents (16 different reagents and combinations thereof), as well as sorting droplets with different chemical properties, on the basis of fluorescence signals. The Braille Display platform is compact, versatile and cost efficient (only ~US$1,000 on top of a standard droplet microfluidics setup). The procedure includes manufacturing of microfluidic chips, assembly of custom hardware, co-encapsulation of cells and drugs into droplets, fluorescence detection of readout signals and data analysis using shared, freely available LabVIEW and Python packages. As a first application, we demonstrate the complete workflow for screening cancer cell drug sensitivities toward 74 conditions. Furthermore, we describe here an assay enabling the normalization of the observed drug sensitivity to the number of cancer cells per droplet, which additionally increases the robustness of the system. As a second application, we also demonstrate the sorting of droplets according to enzymatic activity. The drug screening application can be completed within 2 d; droplet sorting takes ~1 d; and all preparatory steps for manufacturing molds, chips and setting up the Braille controller can be accomplished within 1 week.


Assuntos
Bioensaio , Microfluídica , Microfluídica/métodos , Movimento Celular
3.
Small ; 16(9): e1904321, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31747127

RESUMO

Tailoring patient-specific treatments for cancer is necessary in order to achieve optimal results but requires new diagnostic approaches at affordable prices. Microfluidics has immense potential to provide solutions for this, as it enables the processing of samples that are not available in large quantities (e.g., cells from patient biopsies), is cost efficient, provides a high level of automation, and allows the set-up of complex models for cancer studies. In this review, individual solutions in the fields of genetics, circulating tumor cell monitoring, biomarker analysis, phenotypic drug sensitivity tests, and systems providing controlled environments for disease modeling are discussed. An overview on how these early stage achievements can be combined or developed further is showcased, and the required translational steps before microfluidics becomes a routine tool for clinical applications are critically discussed.


Assuntos
Microfluídica , Neoplasias , Medicina de Precisão , Humanos , Monitorização Fisiológica , Neoplasias/terapia , Células Neoplásicas Circulantes , Medicina de Precisão/instrumentação , Medicina de Precisão/métodos , Medicina de Precisão/tendências
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