RESUMO
Synthetic biologists aim to design biological systems for a variety of industrial and medical applications, ranging from biofuel to drug production. Synthetic gene circuits regulating efflux pump protein expression can achieve this by driving desired substrates such as biofuels, pharmaceuticals, or other chemicals out of the cell in a precisely controlled manner. However, efflux pumps may introduce implicit negative feedback by pumping out intracellular inducer molecules that control gene circuits, which then can alter gene circuit function. Therefore, synthetic gene circuits must be carefully designed and constructed for precise efflux control. Here, we provide protocols for quantitatively modeling and building synthetic gene constructs for efflux pump regulation.
Assuntos
Redes Reguladoras de Genes/genética , Proteínas de Membrana Transportadoras/genética , Biocombustíveis , Genes Sintéticos/genética , Biologia Sintética/métodosRESUMO
Synthetic biology aims to design new biological systems for predefined purposes, such as the controlled secretion of biofuels, pharmaceuticals, or other chemicals. Synthetic gene circuits regulating an efflux pump from the ATP-binding cassette (ABC) protein family could achieve this. However, ABC efflux pumps can also drive out intracellular inducer molecules that control the gene circuits. This will introduce an implicit feedback that could alter gene circuit function in ways that are poorly understood. Here, we used two synthetic gene circuits inducible by tetracycline family molecules to regulate the expression of a yeast ABC pump (Pdr5p) that pumps out the inducer. Pdr5p altered the dose-responses of the original gene circuits substantially in Saccharomyces cerevisiae. While one aspect of the change could be attributed to the efflux pumping function of Pdr5p, another aspect remained unexplained. Quantitative modeling indicated that reduced regulator gene expression in addition to efflux pump function could fully explain the altered dose-responses. These predictions were validated experimentally. Overall, we highlight how efflux pumps can alter gene circuit dynamics and demonstrate the utility of mathematical modeling in understanding synthetic gene circuit function in new circumstances.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Redes Reguladoras de Genes , Genes Sintéticos , Modelos Teóricos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Relação Dose-Resposta a Droga , Doxiciclina/administração & dosagem , Doxiciclina/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Tetraciclina/farmacologiaRESUMO
Previous studies have found the close correlation between epitope density and epitope-specific response, which have shown that high epitope density in a single recombinant protein molecule significantly enhances the humoral response and protective immunity. However, it has not been determined whether this kind of high epitope density could also significantly influence T cell response. Based on this, a series of recombinant DNA and proteins were designed and prepared. Each molecule consists of various copy numbers of the V3 CTL epitope on HIV-1 gp120 (one, two, four and eight copies). Our results show clearly that different V3-epitope densities in just one single DNA or protein molecules have respectively different effects on the number and activity of both primary and memory T cells. Interestingly, this effect is more complex than that on the B cells: epitope density in one plasmid or protein antigen affects the number, not the cytotoxic avidity, of primary CD8+ T cells, but affects both the number and cytotoxic avidity of memory CD8+ T cells. It indicates epitope density in the antigen is an important consideration to optimize T cell response induction and may facilitate the development of effective T cell-based anti-virus vaccines.
Assuntos
Vacinas contra a AIDS/imunologia , Epitopos de Linfócito T/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Memória Imunológica/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Testes Imunológicos de Citotoxicidade , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/imunologiaRESUMO
A novel method for the determination of peroxynitrite using folic acid as a fluorescent probe is described. The method is based on the oxidation of the reduced, low-fluorescent folic acid by peroxynitrite to produce a high-fluorescent emission product. The fluorescence increase is linearly related to the concentration of peroxynitrite in the range of 3x10(-8) to 5.0x10(-6)molL(-1) with a correlation coefficient of 0.998, and the detection limit is 1x10(-8)molL(-1). Interferences from some metal ions normally seen in biological samples, and also some anions structurally similar to peroxynitrite were studied. The optimal conditions for the detection of peroxynitrite were evaluated.