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Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803.

Sakai Y, Abe K, Nakashima S, Ellinger JJ, Ferri S, Sode K, Ikebukuro K - Microbiologyopen (2015)

Bottom Line: Here, we demonstrated that the scaffold sequence fused to the riboregulators improved their gene regulation ability in Synechocystis sp.PCC 6803.The scaffold sequence derived from natural E. coli noncoding small RNAs is effective for designing RNA-based genetic tools and scaffold-fused riboregulators are a strong RNA-tool to regulate gene expression in cyanobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

No MeSH data available.


Related in: MedlinePlus

Synechocystis sp. PCC 6803 expressing Escherichia coli-derived Hfq. (A) Schematic representation of the integration of E. coli-derived Hfq via homologous recombination. His6-tag-fused hfq was regulated by rbcL promoter and integrated into the neutral site of Synechocystis sp. PCC 6803 genomic DNA. (B) The expression of His6-tag-fused Hfq in Synechocystis sp. PCC 6803 Ecohfq::kan cells was confirmed by western blot analysis using anti-His6-tag antibody. (C) The growth curve analysis of Synechocystis sp. PCC 6803 cells integrated with the kan cassette (Synechocystis sp. PCC 6803 kan), and Synechocystis sp. PCC 6803 Ecohfq::kan. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate.
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fig03: Synechocystis sp. PCC 6803 expressing Escherichia coli-derived Hfq. (A) Schematic representation of the integration of E. coli-derived Hfq via homologous recombination. His6-tag-fused hfq was regulated by rbcL promoter and integrated into the neutral site of Synechocystis sp. PCC 6803 genomic DNA. (B) The expression of His6-tag-fused Hfq in Synechocystis sp. PCC 6803 Ecohfq::kan cells was confirmed by western blot analysis using anti-His6-tag antibody. (C) The growth curve analysis of Synechocystis sp. PCC 6803 cells integrated with the kan cassette (Synechocystis sp. PCC 6803 kan), and Synechocystis sp. PCC 6803 Ecohfq::kan. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate.

Mentions: To further improve the gene regulation ability of scaffold-fused taR*2, we aimed to utilize the Hfq protein in Synechocystis. In contrast to E. coli-derived Hfq, the Hfq-dependent natural sRNAs or the preferable binding sequence of Synechocystis-derived Hfq has not been well investigated. Therefore, we chose to introduce and express E. coli-derived Hfq in Synechocystis. Since the scaffold-fused taRNAs had stronger function and were more stable than the taRNAs without the scaffold sequence in E. coli, likely due to the Hfq binding, the scaffold-fused taR*2 might show improved gene regulation ability in Synechocystis expressing E. coli-derived Hfq. Therefore, we integrated E. coli-derived Hfq into the genomic DNA of Synechocystis via homologous recombination. The hfq gene was cloned from E. coli K-12 genomic DNA and a his6-tag was fused to the C-terminus and the expression was regulated by the rbcL promoter (PrbcL). This E. coli-derived Hfq expressing cassette was inserted into the integration vector, pSTVISK, which enables integration of the target DNA sequence and a kanamycin cassette into slr0168, a well-known neutral site (Fig.3A) (Williams 1988). The Synechocystis strain into which E. coli-derived Hfq was integrated, termed Synechocystis Ecohfq::kan, was selected on BG-11 agar plates containing kanamycin at a final concentration of 20 μg/mL. Expression of E. coli-derived Hfq was confirmed by western blot analysis using anti-His6-tag antibody (Fig.3B). However, the expression of E. coli-derived Hfq reduced the growth rate slightly (Fig.3C). This could be due to the variation in the level of endogenous RNA caused by the binding of E. coli-derived Hfq. It has been shown that the binding affinity of E. coli-derived Hfq against their preferable RNA sequence is strong (KD ∼ 10−8 mol/L) (Link et al. 2009), therefore binding against Synechocystis endogenous RNA could occur. Although, this slower growth rate might be improved by optimizing the expression level of E. coli-derived Hfq.


Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803.

Sakai Y, Abe K, Nakashima S, Ellinger JJ, Ferri S, Sode K, Ikebukuro K - Microbiologyopen (2015)

Synechocystis sp. PCC 6803 expressing Escherichia coli-derived Hfq. (A) Schematic representation of the integration of E. coli-derived Hfq via homologous recombination. His6-tag-fused hfq was regulated by rbcL promoter and integrated into the neutral site of Synechocystis sp. PCC 6803 genomic DNA. (B) The expression of His6-tag-fused Hfq in Synechocystis sp. PCC 6803 Ecohfq::kan cells was confirmed by western blot analysis using anti-His6-tag antibody. (C) The growth curve analysis of Synechocystis sp. PCC 6803 cells integrated with the kan cassette (Synechocystis sp. PCC 6803 kan), and Synechocystis sp. PCC 6803 Ecohfq::kan. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554450&req=5

fig03: Synechocystis sp. PCC 6803 expressing Escherichia coli-derived Hfq. (A) Schematic representation of the integration of E. coli-derived Hfq via homologous recombination. His6-tag-fused hfq was regulated by rbcL promoter and integrated into the neutral site of Synechocystis sp. PCC 6803 genomic DNA. (B) The expression of His6-tag-fused Hfq in Synechocystis sp. PCC 6803 Ecohfq::kan cells was confirmed by western blot analysis using anti-His6-tag antibody. (C) The growth curve analysis of Synechocystis sp. PCC 6803 cells integrated with the kan cassette (Synechocystis sp. PCC 6803 kan), and Synechocystis sp. PCC 6803 Ecohfq::kan. The graphs depict the mean and error bars represent the standard deviation of experiments performed in triplicate.
Mentions: To further improve the gene regulation ability of scaffold-fused taR*2, we aimed to utilize the Hfq protein in Synechocystis. In contrast to E. coli-derived Hfq, the Hfq-dependent natural sRNAs or the preferable binding sequence of Synechocystis-derived Hfq has not been well investigated. Therefore, we chose to introduce and express E. coli-derived Hfq in Synechocystis. Since the scaffold-fused taRNAs had stronger function and were more stable than the taRNAs without the scaffold sequence in E. coli, likely due to the Hfq binding, the scaffold-fused taR*2 might show improved gene regulation ability in Synechocystis expressing E. coli-derived Hfq. Therefore, we integrated E. coli-derived Hfq into the genomic DNA of Synechocystis via homologous recombination. The hfq gene was cloned from E. coli K-12 genomic DNA and a his6-tag was fused to the C-terminus and the expression was regulated by the rbcL promoter (PrbcL). This E. coli-derived Hfq expressing cassette was inserted into the integration vector, pSTVISK, which enables integration of the target DNA sequence and a kanamycin cassette into slr0168, a well-known neutral site (Fig.3A) (Williams 1988). The Synechocystis strain into which E. coli-derived Hfq was integrated, termed Synechocystis Ecohfq::kan, was selected on BG-11 agar plates containing kanamycin at a final concentration of 20 μg/mL. Expression of E. coli-derived Hfq was confirmed by western blot analysis using anti-His6-tag antibody (Fig.3B). However, the expression of E. coli-derived Hfq reduced the growth rate slightly (Fig.3C). This could be due to the variation in the level of endogenous RNA caused by the binding of E. coli-derived Hfq. It has been shown that the binding affinity of E. coli-derived Hfq against their preferable RNA sequence is strong (KD ∼ 10−8 mol/L) (Link et al. 2009), therefore binding against Synechocystis endogenous RNA could occur. Although, this slower growth rate might be improved by optimizing the expression level of E. coli-derived Hfq.

Bottom Line: Here, we demonstrated that the scaffold sequence fused to the riboregulators improved their gene regulation ability in Synechocystis sp.PCC 6803.The scaffold sequence derived from natural E. coli noncoding small RNAs is effective for designing RNA-based genetic tools and scaffold-fused riboregulators are a strong RNA-tool to regulate gene expression in cyanobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

No MeSH data available.


Related in: MedlinePlus