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

Silva-Rocha R, Pontelli MC, Furtado GP, Zaramela LS, Koide T - PLoS ONE (2015)

Bottom Line: 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.This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Immunology-Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.

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.

No MeSH data available.


Related in: MedlinePlus

Construction of H. salinarum strains with chromosomal tags.(A) A suicide plasmid pHsal-S-Lsm::FLAG-tag, harboring a modified flanking region of the Lsm gene to introduce a FLAG-tag epitope, was transformed into H. salinarum. Double recombination events were selected and correct incorporation of the FLAG-tag was checked using primers Pck1 and Pflag. As a control, primers Pck1/Pck2 were used to amplify the whole flanking region of the Lsm coding gene. (B) PCR validation of the FLAG-tag incorporation. Ten (numbered from 1 to 10) independent colonies were selected and screened using primers Pck1/Pflag, which should give rise to an amplification band of ~ 600 bp. The 1.0 kb DNA ladder is shown on line 1, while line 2 shows the amplification control, representing the flanking region obtained using primers Pck1/Pck2.
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pone.0129215.g005: Construction of H. salinarum strains with chromosomal tags.(A) A suicide plasmid pHsal-S-Lsm::FLAG-tag, harboring a modified flanking region of the Lsm gene to introduce a FLAG-tag epitope, was transformed into H. salinarum. Double recombination events were selected and correct incorporation of the FLAG-tag was checked using primers Pck1 and Pflag. As a control, primers Pck1/Pck2 were used to amplify the whole flanking region of the Lsm coding gene. (B) PCR validation of the FLAG-tag incorporation. Ten (numbered from 1 to 10) independent colonies were selected and screened using primers Pck1/Pflag, which should give rise to an amplification band of ~ 600 bp. The 1.0 kb DNA ladder is shown on line 1, while line 2 shows the amplification control, representing the flanking region obtained using primers Pck1/Pck2.

Mentions: To demonstrate the efficacy of the new tools constructed here for genome editing, we used the pHsal-S vector for the generation of H. salinarum strains harboring chromosomal tags in genes of interest. As a proof of concept, we targeted the RNA chaperone Lsm encoded by the gene VNG1496G. We selected the FLAG-tag since it is a widely used and short (8 aa) tag peptide [29], with reduced chances of affecting the structure of the target protein. For the construction of the tagged strain, flanking regions of 500 bp upstream and downstream of the stop codon of the VNG1496G gene were PCR amplified and assembled into a 1.0 kb fragment by recombinant PCR. In this procedure, the homology primers of the recombinant PCR harbor the nucleotide sequence coding the FLAG-tag, allowing the insertion of the tag at the 3’-end of the gene. The final recombinant fragment was then cloned into pHsal-S vector and the resulting plasmid was used to transform H. salinarum. Recombinant strains with the plasmid integrated into the chromosome were selected by plating the transformation in selective media supplemented with mevinolin. After the appearance of colonies in the selective media, a single one was picked and inoculated into liquid media without selective pressure to allow the second event of recombination. After saturation of the liquid culture, cells were plated in solid media with 5-FOA to counter-select non-recombinant strains [13]. Colonies able to grow under these conditions were checked by PCR to verify if the tag was correctly inserted into the chromosome or if the strains were able to revert to the wild-type genotype (Fig 5). As shown in Fig 5B, we detected 7 out of 10 colonies that were positive for the correct incorporation of the FLAG-tag. This result highlights the applicability of the new suicide vector pHsal-S for the generation of stable genotypes in H. salinarum.


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

Silva-Rocha R, Pontelli MC, Furtado GP, Zaramela LS, Koide T - PLoS ONE (2015)

Construction of H. salinarum strains with chromosomal tags.(A) A suicide plasmid pHsal-S-Lsm::FLAG-tag, harboring a modified flanking region of the Lsm gene to introduce a FLAG-tag epitope, was transformed into H. salinarum. Double recombination events were selected and correct incorporation of the FLAG-tag was checked using primers Pck1 and Pflag. As a control, primers Pck1/Pck2 were used to amplify the whole flanking region of the Lsm coding gene. (B) PCR validation of the FLAG-tag incorporation. Ten (numbered from 1 to 10) independent colonies were selected and screened using primers Pck1/Pflag, which should give rise to an amplification band of ~ 600 bp. The 1.0 kb DNA ladder is shown on line 1, while line 2 shows the amplification control, representing the flanking region obtained using primers Pck1/Pck2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465625&req=5

pone.0129215.g005: Construction of H. salinarum strains with chromosomal tags.(A) A suicide plasmid pHsal-S-Lsm::FLAG-tag, harboring a modified flanking region of the Lsm gene to introduce a FLAG-tag epitope, was transformed into H. salinarum. Double recombination events were selected and correct incorporation of the FLAG-tag was checked using primers Pck1 and Pflag. As a control, primers Pck1/Pck2 were used to amplify the whole flanking region of the Lsm coding gene. (B) PCR validation of the FLAG-tag incorporation. Ten (numbered from 1 to 10) independent colonies were selected and screened using primers Pck1/Pflag, which should give rise to an amplification band of ~ 600 bp. The 1.0 kb DNA ladder is shown on line 1, while line 2 shows the amplification control, representing the flanking region obtained using primers Pck1/Pck2.
Mentions: To demonstrate the efficacy of the new tools constructed here for genome editing, we used the pHsal-S vector for the generation of H. salinarum strains harboring chromosomal tags in genes of interest. As a proof of concept, we targeted the RNA chaperone Lsm encoded by the gene VNG1496G. We selected the FLAG-tag since it is a widely used and short (8 aa) tag peptide [29], with reduced chances of affecting the structure of the target protein. For the construction of the tagged strain, flanking regions of 500 bp upstream and downstream of the stop codon of the VNG1496G gene were PCR amplified and assembled into a 1.0 kb fragment by recombinant PCR. In this procedure, the homology primers of the recombinant PCR harbor the nucleotide sequence coding the FLAG-tag, allowing the insertion of the tag at the 3’-end of the gene. The final recombinant fragment was then cloned into pHsal-S vector and the resulting plasmid was used to transform H. salinarum. Recombinant strains with the plasmid integrated into the chromosome were selected by plating the transformation in selective media supplemented with mevinolin. After the appearance of colonies in the selective media, a single one was picked and inoculated into liquid media without selective pressure to allow the second event of recombination. After saturation of the liquid culture, cells were plated in solid media with 5-FOA to counter-select non-recombinant strains [13]. Colonies able to grow under these conditions were checked by PCR to verify if the tag was correctly inserted into the chromosome or if the strains were able to revert to the wild-type genotype (Fig 5). As shown in Fig 5B, we detected 7 out of 10 colonies that were positive for the correct incorporation of the FLAG-tag. This result highlights the applicability of the new suicide vector pHsal-S for the generation of stable genotypes in H. salinarum.

Bottom Line: 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.This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Immunology-Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.

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.

No MeSH data available.


Related in: MedlinePlus