Stable nuclear transformation of the diatom Phaeodactylum tricornutum.
TL;DR: This work demonstrates that heterologous genes can be readily expressed in P. tricornutum and provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.
Abstract: A nuclear transformation system has been developed for the diatomPhaeodactylum tricornutum using microparticle bombardment to introduce thesh ble gene fromStreptoalloteichus hindustanus into cells. Thesh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from theP. tricornutum fcp genes were used to drive expression ofsh ble. Between 10–100 transformants were recovered/108 cells. Transformants were able to grow on at least 500 µg/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations thesh ble gene was present in 1–3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1–2.0 µg/mg total protein). Thecat reporter gene fused to afcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed inP. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.
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TL;DR: A review of second generation biodiesel production systems using microalgae can be found in this paper, where the main advantages of second-generation microalgal systems are that they: (1) have a higher photon conversion efficiency (as evidenced by increased biomass yields per hectare): (2) can be harvested batch-wise nearly all-year-round, providing a reliable and continuous supply of oil: (3) can utilize salt and waste water streams, thereby greatly reducing freshwater use: (4) can couple CO2-neutral fuel production with CO2 sequestration: (
Abstract: The use of fossil fuels is now widely accepted as unsustainable due to depleting resources and the accumulation of greenhouse gases in the environment that have already exceeded the “dangerously high” threshold of 450 ppm CO2-e. To achieve environmental and economic sustainability, fuel production processes are required that are not only renewable, but also capable of sequestering atmospheric CO2. Currently, nearly all renewable energy sources (e.g. hydroelectric, solar, wind, tidal, geothermal) target the electricity market, while fuels make up a much larger share of the global energy demand (∼66%). Biofuels are therefore rapidly being developed. Second generation microalgal systems have the advantage that they can produce a wide range of feedstocks for the production of biodiesel, bioethanol, biomethane and biohydrogen. Biodiesel is currently produced from oil synthesized by conventional fuel crops that harvest the sun’s energy and store it as chemical energy. This presents a route for renewable and carbon-neutral fuel production. However, current supplies from oil crops and animal fats account for only approximately 0.3% of the current demand for transport fuels. Increasing biofuel production on arable land could have severe consequences for global food supply. In contrast, producing biodiesel from algae is widely regarded as one of the most efficient ways of generating biofuels and also appears to represent the only current renewable source of oil that could meet the global demand for transport fuels. The main advantages of second generation microalgal systems are that they: (1) Have a higher photon conversion efficiency (as evidenced by increased biomass yields per hectare): (2) Can be harvested batch-wise nearly all-year-round, providing a reliable and continuous supply of oil: (3) Can utilize salt and waste water streams, thereby greatly reducing freshwater use: (4) Can couple CO2-neutral fuel production with CO2 sequestration: (5) Produce non-toxic and highly biodegradable biofuels. Current limitations exist mainly in the harvesting process and in the supply of CO2 for high efficiency production. This review provides a brief overview of second generation biodiesel production systems using microalgae.
2,254 citations
Cites methods from "Stable nuclear transformation of th..."
...This technique involves bombardment with DNA-coated microprojectiles and has been successfully used for a variety of algae including green algae and diatoms [8, 94], and is also the method of choice for chloroplast or mitochondrial genome transformation [143]....
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TL;DR: Potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes are focused on.
Abstract: There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.
1,079 citations
Cites background or methods from "Stable nuclear transformation of th..."
...tricornutum (2, 210, 211), Navicula saprophila (45), Cylindrotheca fusiformis (52, 148), Cyclotella cryptica (45), and Thalassiosira weissflogii (51); and phaeophytes, such as Laminaria japonica (150) and Undaria pinnatifada (151)....
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...This problem could be circumvented by the introduction of an AGPase that does not require 3-PGA, such as the Mos(1-198)/SH2 AGPase, which still has activity even without the presence of an activator (14)....
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...Several different antibiotic resistance genes have been used successfully for microalgal transformant selection, including bleomycin (2, 52, 56, 104, 210), spectinomycin (19, 42), streptomycin (42), paromomycin (81, 173), nourseothricin (210), G418 (45, 148, 210), hygromycin (12), chloramphenicol (184), and others....
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...A variety of transformation methods have been used to transfer DNA into microalgal cells, including agitation in the presence of glass beads or silicon carbide whiskers (44, 87, 119), electroporation (21, 22, 26, 108, 170, 181, 184), biolistic microparticle bombardment (2, 45, 49, 51, 52, 81, 83, 88, 183, 187, 210, 211), and Agrobacterium tumefaciens-mediated gene transfer (23, 93)....
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...the introduction of an AGPase that does not require 3-PGA, such as the Mos(1-198)/SH2 AGPase, which still has activity even without the presence of an activator (14)....
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TL;DR: The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research.
Abstract: Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research.
Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.
834 citations
01 Sep 2010
TL;DR: This article attempts to elucidate the major challenges to economic algal biofuels at scale, and improves the focus of the scientific community to address these challenges and move algalBiofuels from promise to reality.
Abstract: Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality.
680 citations
Cites background from "Stable nuclear transformation of th..."
...Whole-genome sequences have been released for two species, Thalassiosira pseudonana and Phaeodactylum tricornutum, and recent work has shown stable nuclear transformation of these two species and a few other diatoms [91–94]....
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TL;DR: In this article, the authors reviewed the potential of microalgae and macroalgae for the production of bio-diesel and micro-algae as a promising alternative source to the conventional feedstocks for the third generation biofuel production.
Abstract: Due to diminishing petroleum reserves and deleterious environmental consequences of exhaust gases from fossil-based fuels, research on renewable and environment friendly fuels has received a lot of impetus in the recent years. However, the availability of the non-edible crops serve as the sources for biofuel production are limited and economically not feasible. Algae are a promising alternative source to the conventional feedstocks for the third generation biofuel production. There has been a considerable discussion in the recent years about the potential of microalgae for the production of biofuels, but there may be other more readily exploitable commercial opportunities for macroalgae and microalgae. This review, briefly describes the biofuels conversion technologies for both macroalgae and microalgae. The gasification process produces combustible gases such as H2, CH4, CO2 and ammonia, whereas, the product of pyrolysis is bio-oil. The fermentation product of algae is ethanol, that can be used as a direct fuel or as a gasohol. Hydrogen can be obtained from the photobiological process of algal biomass. In transesterification process, algae oil is converted into biodiesel, which is quite similar to those of conventional diesel and it can be blended with the petroleum diesel. This study, also reviewed the production of high value byproducts from macroalgae and microalgae and their commercial applications. Algae as a potential renewable resource is not only used for biofuels but also for human health, animal and aquatic nutrition, environmental applications such as CO2 mitigation, wastewater treatment, biofertilizer, high-value compounds, synthesis of pigments and stable isotope biochemicals. This review is mainly an attempt, to investigate the biorefinery concept applied on the algal technology, for the synthesis of novel bioproducts to improve the algal biofuels as even more diversified and economically competitive. The employment of a high-value, co-product strategy through the integrated biorefinery approach is expected to significantly enhance the overall commercial implementation of the biofuel from the algal technology.
547 citations
References
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TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
"Stable nuclear transformation of th..." refers methods in this paper
...Protein samples (10 gg total protein) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (15% acrylamide) utilizing the Laemmli (1970) buffer system....
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
Journal Article•
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
01 Jan 1975
TL;DR: The methods suffice for the most fastidious algae now routinely cultivable, and simplifications indicated for less demanding species are easily made; for example, omission of silicate for plants other than diatoms.
Abstract: These pages describe relatively simple and reliable methods for the culture of marine phytoplankton species useful for feeding marine invertebrates. The methods suffice for the most fastidious algae now routinely cultivable, and simplifications indicated for less demanding species are easily made; for example, omission of silicate for plants other than diatoms. Certain modifications of techniques, ancillary methods, and precautions will be treated briefly because questions often arise concerning them, but documentation will be minimal and hopefully restricted to publications readily available.
5,000 citations
"Stable nuclear transformation of th..." refers methods in this paper
...Liquid cultures grown in f/2 medium (Guillard 1975) were bubbled with air containing 1% CO2....
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3,224 citations