Desenvolvimento e padronização de um sistema de autoindução da expressão gênica em Bacillus subtilis
Carregando...
Data
2019-07-29
Autores
Corrêa, Graciely Gomes
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os métodos de indução da expressão gênica disponíveis para linhagens bacterianas envolvem a adição de compostos indutores ao meio de cultura (por exemplo, isopropil β-D-1-tiogalactopiranosideo, xilose e arabinose), o que é indesejável para linhagens industriais, pois encarece o processo produtivo. Já a utilização da expressão constitutiva, alternativa à indução, pode ocasionar stress metabólico durante a fase lag de crescimento quando são utilizados promotores fortes. O objetivo do trabalho foi construir e padronizar um novo modelo de indução da expressão gênica para linhagens bacterianas industriais. O novo modelo de autoindução baseado no sistema de quorum-sensing bacteriano, permitindo que a célula se automonitore e induza a expressão gênica durante a fase exponencial de crescimento, eliminando assim não só a necessidade de adição de composto indutor como a necessidade de monitoramento da densidade celular pré-indução. Realizou-se amplificação e clonagem dos genes luxR e luxI, com e sem caudas de histidina, e suas respectivas sequências regulatórias de Aliivibrio fischeri, em plasmídeo contendo os genes responsáveis pela bioluminescência ou fluorescência com códons otimizados para Bacillus subtilis. Em seguida, foi realizada transformação e a integração do plasmídeo no cromossomo de B. subtilis. A funcionalidade do sistema foi avaliada em diferentes etapas de crescimento microbiano com o auxílio de um leitor de microplacas durante intervalos regulares. O sistema de autoindução mostrou-se funcional em B. subtilis, com indução acentuada da expressão do gene repórter, disposto downstream ao promotor Plux, durante a fase exponencial de crescimento. Verificou-se ainda que a inserção do sistema baseado no quorum-sensing não afetou o crescimento do microrganismo significativamente. Foram desenvolvidas e testadas dez otimizações do sistema inicial, com alterações na sequência do promotor Plux. Através dessas otimizações, construímos linhagens com diferentes níveis de expressão do sistema de autoindução e força do promotor variável. Ao caracterizar os sistemas, foi constatado alto nível de modularidade, podendo ser utilizados em diferentes linhagens de B. subtilis, diferentes contextos genéticos e para a produção de diferentes produtos. O modelo de indução desenvolvido se mostrou funcional e modular e poderá ser adaptado a diferentes espécies e compostos, configurando uma nova ferramenta para a biologia sintética.
Current methods available for the autoinduction of gene expression in genetically engineered bacterial strains require addition of inducing compounds to the culture medium (e.g. Isopropyl β-D-1-thiogalactopyranoside, xylose, and arabinose), which is undesirable for industrial strains due to additional costs to the production process. Alternatively, constitutive gene expression is employed. However, the later can possibly cause metabolic stress during the lag growth phase if strong promoters are employed. The objective of this work was to construct and to standardize a new model for induction of gene expression in industrial bacterial strains. The new model is based on an autoinduction process triggered by the bacterial quorum-sensing system. It allows the cell to monitor itself and induce its own gene expression during the exponential growth phase, thereby eliminating both the need for an external inducing compound and the need for monitoring pre-inducing cell density. Bacterial cultures were grown in rich media, supplemented or not with antibiotics. Amplification and cloning of luxR and luxI genes, with and without histidine tags, and their respective regulatory sequences of Aliivibrio fischeri, were performed on a plasmid containing the genes responsible for bioluminescence or fluorescence with codons optimized for Bacillus subtilis. Next, transformation and integration of the plasmid into the B. subtilis chromosome were performed. The functionality of the system was evaluated at different stages of microbial growth, by luminescence or fluorescence measurement at regular intervals, using a microplate reader. The autoinduction system promoted accentuated induction of reporter gene expression located downstream of the Plux promoter during the exponential growth phase. It was also found that the insertion of the quorum-sensing system did not significantly affect the microorganism growth. Ten variations of the autoinduction system were generated and tested, which consisted of optimizations of the Plux promoter. Through the optimizations, we constructed strains showing a range of gene expression fold change and an array of promoter strengths. The constructed autoinduction systems show high level modularity, as they are functional in different strains of B. subtilis, in different genetic contexts and can efficiently control the production of different products. The developed induction model proved to be functional and modular, and it could be adapted to different species and compounds, configuring a new tool for synthetic biology.
Current methods available for the autoinduction of gene expression in genetically engineered bacterial strains require addition of inducing compounds to the culture medium (e.g. Isopropyl β-D-1-thiogalactopyranoside, xylose, and arabinose), which is undesirable for industrial strains due to additional costs to the production process. Alternatively, constitutive gene expression is employed. However, the later can possibly cause metabolic stress during the lag growth phase if strong promoters are employed. The objective of this work was to construct and to standardize a new model for induction of gene expression in industrial bacterial strains. The new model is based on an autoinduction process triggered by the bacterial quorum-sensing system. It allows the cell to monitor itself and induce its own gene expression during the exponential growth phase, thereby eliminating both the need for an external inducing compound and the need for monitoring pre-inducing cell density. Bacterial cultures were grown in rich media, supplemented or not with antibiotics. Amplification and cloning of luxR and luxI genes, with and without histidine tags, and their respective regulatory sequences of Aliivibrio fischeri, were performed on a plasmid containing the genes responsible for bioluminescence or fluorescence with codons optimized for Bacillus subtilis. Next, transformation and integration of the plasmid into the B. subtilis chromosome were performed. The functionality of the system was evaluated at different stages of microbial growth, by luminescence or fluorescence measurement at regular intervals, using a microplate reader. The autoinduction system promoted accentuated induction of reporter gene expression located downstream of the Plux promoter during the exponential growth phase. It was also found that the insertion of the quorum-sensing system did not significantly affect the microorganism growth. Ten variations of the autoinduction system were generated and tested, which consisted of optimizations of the Plux promoter. Through the optimizations, we constructed strains showing a range of gene expression fold change and an array of promoter strengths. The constructed autoinduction systems show high level modularity, as they are functional in different strains of B. subtilis, in different genetic contexts and can efficiently control the production of different products. The developed induction model proved to be functional and modular, and it could be adapted to different species and compounds, configuring a new tool for synthetic biology.
Descrição
Palavras-chave
Quorum-sensing, sistema lux, B. subtilis, biologia sintética, expressão gênica, Quorum-sensing, lux system, B. subtilis, synthetic biology, gene expression