RESUMO
We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Engenharia Metabólica/métodos , Percepção de Quorum/genética , Transdução de Sinais/genética , Acil-Butirolactonas/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Homosserina/análogos & derivados , Homosserina/metabolismo , Humanos , Lactonas/metabolismo , Microrganismos Geneticamente Modificados , Plasmídeos/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
We generated "sentinel" bacteria that respond to the biomarker nitric oxide (NO) and produce a homogeneous and strong fluorescent response. Our dual-plasmid system consists of a signal "relay" vector that employs an NO-responsive promoter that amplifies the native signal (via expression of T7 Polymerase (T7Pol)) to a second vector responsible for GFP expression. Importantly, to achieve an optimal "sentinel" response, we developed strategies that balance the transcriptional load within cells by altering (i) translation and (ii) activity of the T7Pol. Our optimized genetic circuitry was then used to transform commensal E. coli Nissle, as a proof-of-concept toward an ingestible cell-based sensor for Crohn's disease (CD) that, in turn, is marked by elevated levels of intestinal NO. Thus, the "biosensors" demonstrated here may serve as a simple diagnostic tool, contrasting the standard of care including colonoscopies or biopsies.
Assuntos
Óxido Nítrico/metabolismo , Animais , Biomarcadores , Técnicas Biossensoriais , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Escherichia coli/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Regiões Promotoras Genéticas/genética , Biologia Sintética/métodosRESUMO
LipL32 is a major surface protein that is expressed during infection by pathogenic Leptospira. Here, the crystallization of recombinant LipL32(21-272), which corresponds to the mature LipL32 protein minus its N-terminal lipid-anchored cysteine residue, is described. Selenomethionine-labelled LipL32(21-272) crystals diffracted to 2.25 A resolution at a synchrotron source. The space group was P3(1)21 or P3(2)21 and the unit-cell parameters were a = b = 126.7, c = 96.0 A.