RESUMO
Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.
Assuntos
Actinobacteria/genética , Firmicutes/genética , Ensaios de Triagem em Larga Escala , Proteobactérias/genética , Frações Subcelulares/metabolismo , Transcrição Gênica , Actinobacteria/química , Actinobacteria/metabolismo , Firmicutes/química , Firmicutes/metabolismo , Biblioteca Gênica , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteobactérias/química , Proteobactérias/metabolismo , Frações Subcelulares/química , Biologia Sintética/métodosRESUMO
Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract remains the major cause of morbidity and nonrelapse mortality after BM transplantation (BMT). The Paneth cell protein regenerating islet-derived 3α (REG3α) is a biomarker specific for GI GVHD. REG3α serum levels rose in the systematic circulation as GVHD progressively destroyed Paneth cells and reduced GI epithelial barrier function. Paradoxically, GVHD suppressed intestinal REG3γ (the mouse homolog of human REG3α), and the absence of REG3γ in BMT recipients intensified GVHD but did not change the composition of the microbiome. IL-22 administration restored REG3γ production and prevented apoptosis of both intestinal stem cells (ISCs) and Paneth cells, but this protection was completely abrogated in Reg3g-/- mice. In vitro, addition of REG3α reduced the apoptosis of colonic cell lines. Strategies that increase intestinal REG3α/γ to promote crypt regeneration may offer a novel, nonimmunosuppressive approach for GVHD and perhaps for other diseases involving the ISC niche, such as inflammatory bowel disease.
Assuntos
Apoptose , Transplante de Medula Óssea , Colo/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Proteínas Associadas a Pancreatite/metabolismo , Celulas de Paneth/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular/genética , Colo/patologia , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas a Pancreatite/genética , Celulas de Paneth/patologia , Estudos Prospectivos , Transplante HomólogoRESUMO
Advances in synthetic biology to build microbes with defined and controllable properties are enabling new approaches to design and program multispecies communities. This emerging field of synthetic ecology will be important for many areas of biotechnology, bioenergy and bioremediation. This endeavor draws upon knowledge from synthetic biology, systems biology, microbial ecology and evolution. Fully realizing the potential of this discipline requires the development of new strategies to control the intercellular interactions, spatiotemporal coordination, robustness, stability and biocontainment of synthetic microbial communities. Here, we review recent experimental, analytical and computational advances to study and build multi-species microbial communities with defined functions and behavior for various applications. We also highlight outstanding challenges and future directions to advance this field.