| Full text | |
| Author(s): |
Silva-Rocha, Rafael
[1]
;
Pontelli, Marjorie Cornejo
[1]
;
Furtado, Gilvan Pessoa
[1]
;
Zaramela, Livia Soares
[1]
;
Koide, Tie
[1]
Total Authors: 5
|
| Affiliation: | [1] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Ribeirao Preto, SP - Brazil
Total Affiliations: 1
|
| Document type: | Journal article |
| Source: | PLoS One; v. 10, n. 6 JUN 10 2015. |
| Web of Science Citations: | 1 |
| 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. (AU) | |
| FAPESP's process: | 13/04125-2 - New genetic tools for Archaea: functional characterization and modeling of the non-coding RNA regulatory network of Halobacterium salinarum |
| Grantee: | Rafael Silva Rocha |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 11/07487-7 - Identification and characterization of Lsm-binding non-coding RNAs in the extremophile Halobacterium salinarum |
| Grantee: | Lívia Soares Zaramela |
| Support Opportunities: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 13/23712-6 - Identification of small proteins and peptides in extremophile Halobacterium salinarum: discriminating of non-coding RNAs and small ORFs |
| Grantee: | Gilvan Pessoa Furtado |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 09/09532-0 - Systems biology of the extremophile Halobacterium salinarum: role of the non-coding RNAs in the global gene regulatory network model |
| Grantee: | Tie Koide |
| Support Opportunities: | Research Grants - Young Investigators Grants |