Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum

Autor(es)

Silva-Rocha, Rafael ^[1] ; Pontelli, Marjorie Cornejo ^[1] ; Furtado, Gilvan Pessoa ^[1] ; Zaramela, Livia Soares ^[1] ; Koide, Tie ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PLoS One; v. 10, n. 6 JUN 10 2015.
• Citações Web of Science: 1

Resumo

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli's specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea. (AU)

• Processo FAPESP: 13/04125-2 - Novas ferramentas genéticas para Archaea: validação funcional e modelagem da rede regulatória de RNA não codificantes de Halobacterium salinarum
• Beneficiário: Rafael Silva Rocha
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 11/07487-7 - Identificação e caracterização de RNAs não codificantes ligantes a Lsm no extremófilo Halobacterium salinarum
• Beneficiário: Lívia Soares Zaramela
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/23712-6 - Identificação de pequenas proteínas e peptídeos no extremófilo Halobacterium salinarum: discriminando RNAs não-codificantes de pequenas ORFs
• Beneficiário: Gilvan Pessoa Furtado
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 09/09532-0 - Biologia sistêmica do extremófilo Halobacterium salinarum: contribuição dos RNAs não-codificantes ao modelo global de regulação gênica
• Beneficiário: Tie Koide
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores