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

PLoS One. 2015 Jun 10;10(6):e0129215. doi: 10.1371/journal.pone.0129215. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular / methods
  • DNA, Archaeal
  • Genetic Engineering / methods*
  • Genetic Vectors / genetics*
  • Genome Size
  • Halobacterium salinarum / genetics*
  • Models, Genetic

Substances

  • DNA, Archaeal

Grants and funding

This work was supported by the Young Researcher Program of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - http://fapesp.br/en/) [09/09532-0 to TK], Edital Universal do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - http://www.cnpq.br/) [473660/2013-0 to TK, 470120/2009-6 to TK], Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA); Núcleo de Apoio a Pesquisa em Biologia Computacional e Genômica (NAP-NUBIC) from Universidade de São Paulo; fellowships from FAPESP [13/04125-2 to RSR, 11/07487-7 to LSZ, 13/23712-6 to GPF] and CAPES [to MCP].