Simple, Rapid and Cost-Effective Method for High Quality Nucleic Acids Extraction from Different Strains of Botryococcus braunii

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics

Determination of Cyanobacterial Diversity during Algal Blooms in Daechung Reservoir, Korea, on the Basis of cpcBA Intergenic Spacer Region Analysis

The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes

Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina

Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA) PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals

RT-qPCR of <i>B. braunii</i> NIES 836 18 S rRNA gene of 213 bp (A), <i>rBcL</i> gene of 343 bp (B), 18 S rRNA gene of 431 bp (C), and 18 S rRNA gene of 527 bp (D).

<p>Closed square; P kit, open square; Q kit, closed diamond; F&T method, open diamond; BPC method, closed triangle; EMNE method.</p

Comparison of quality and quantity of DNA samples extracted from B. <i>braunii</i> using different methods.

a<p>The DNA purity was estimated by calculating the ratio between the absorbance at 260 nm and 280 nm.</p>b<p>The DNA concentration was quantified using a spectrophotometer at 260 nm.</p>c<p>The cultured cells were harvested from culture of 50 ml (commercial kit), 3 ml (BPC & F&T methods), and 1 ml (EMNE method for <i>B. braunii</i>), respectively.</p>d<p>The DNA yield was estimated by calculating the DNA concentration between the dry cell weight and total DNA.</p><p>CCALA, Culture Collection of Autotrophic Organisms; NIES, National Institute for Environmental Studies; UTEX, the Culture Collection of Algae at the University of Texas.</p

Boletín de Segovia: Número 78 - 1923 junio 29

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Gel electrophoresis of total RNA extracted from <i>Botryococcus</i> sp by EMNE (A), F&T (B), and BPC method (C).

<p>RNA samples were electrophoresed on a 1.6% agarose gel in 0.5× TAE buffer. Lane M, 1 Kb ladder; lane 1, <i>Botryococcus braunii</i> CCALA 776; lane 2, <i>B. braunii</i> CCALA 777; lane 3, <i>B. braunii</i> CCALA 778; lane 4, <i>B. braunii</i> CCALA 779; lane 5, <i>B. braunii</i> UTEX 572.</p

Cost Effectiveness Analysis of different methods used for DNA extraction.

<p>Baseline scores and relative scores for each parameter for different methods have been depicted.</p

Summary of nucleic acids extraction from eukaryotic microalgae using EMNE method.

<p>Summary of nucleic acids extraction from eukaryotic microalgae using EMNE method.</p

Comparison of yield and purity of RNA samples prepared by EMNE, F&T, and BPC method.

a<p>The RNA purity was estimated by calculating the ratio between the absorbance at 260 nm and 280 nm.</p>b<p>The RNA concentration was quantified using a spectrophotometer at 260 nm.</p>c<p>The cultured cells were harvested from culture of 3 ml (BPC & F&T methods) and 1 ml (EMNE method for <i>B. braunii</i>), respectively.</p><p>CCALA, Culture Collection of Autotrophic Organisms; UTEX, the Culture Collection of Algae at the University of Texas at Austin.</p