DOCSLIB.ORG

Crystallization of RNA and RNA–Protein Complexes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crystallization of RNA and RNA–Protein Complexes Methods 34 (2004) 408–414 www.elsevier.com/locate/ymeth Crystallization of RNA and RNA–protein complexes Ailong Kea and Jennifer A. Doudnaa,b,¤ a Department of Molecular and Cell Biology and Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA b Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA Accepted 24 March 2004 Abstract RNA plays a direct role in a variety of cellular activities, and in many cases its biological function is conferred by the RNA three- dimensional structure. X-ray crystallography is the method of choice for determining high resolution structures of large RNA molecules, and can also be used to compare related RNAs and identify conformational changes that may accompany biochemical activity. However, crystallization remains the rate-limiting step in RNA structure determination due to the diYculty in obtaining well-ordered crystals for X-ray diVraction analysis. Several approaches to sample preparation, crystallization, and crystal handling are presented that have been used successfully in the structure determination of RNA and RNA–protein complexes in our labora- tory, and should be generally applicable to RNAs in other systems. 2004 Elsevier Inc. All rights reserved. Keywords: RNA; Ribozyme; Crystallization; RNP 1. Introduction the past few years have revealed many of the principles that enable RNA helices to pack into compact three- The explosion of interest in RNA and RNA–protein dimensional structures that create catalytic centers and complexes in recent years stems from the discovery that ligand binding sites. Nonetheless, much remains to be RNA plays much more varied roles in biology than had learned about RNA that can only be revealed in detail previously been suspected. In addition to its long-recog- by X-ray crystal structure determination. This review nized participation in all aspects of protein biosynthesis, outlines a variety of methods used to prepare and crys- structured RNA molecules are also central to the activi- tallize RNA samples and optimize crystals for X-ray ties of ribonucleoprotein complexes responsible for telo- diVraction analysis, with examples from experience in mere replication, X-chromosome inactivation, protein our own laboratory. traYcking, and RNA splicing and processing. In addi- tion, RNA interference involves hundreds of small RNA oligonucleotides that are processed from larger precur- 2. Principles of RNA crystallization sors and inactivate speciWc messenger RNAs by mecha- nisms whose details have yet to be elucidated. Along Crystallization is predictably the least predictable with the increased interest in RNA biology has come the aspect of a structure determination project. Many fac- recognition that understanding RNA activities will tors aVect the inherent crystallizability of an RNA sam- require knowledge of the structures and conformational ple, including purity, conformational homogeneity, dynamics of these molecules. The crystal structures of molecular surface area, and available sites for inter- ribozymes and of the ribosome and its subunits solved in molecular contacts, ligand binding, and structural dynamics. A crystal is a well-ordered three-dimensional ¤ Corresponding author. Fax: 1-510-643-0080. array of molecules held together by non-covalent inter- E-mail address: [email protected] (J.A. Doudna). actions called “crystal contacts.” In general, crystals of 1046-2023/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2004.03.027 A. Ke, J.A. Doudna / Methods 34 (2004) 408–414 409 nucleic acids and proteins can be grown by slow, con- resolution for the Ffh M-domain bound to 4.5S RNA trolled precipitation from aqueous solution under [9]. Another successful approach involves engineering non-denaturing conditions. Among the various crystalli- the U1A binding site sequence into non-essential regions zation techniques available, the most popular are hang- of the RNA, and crystallizing the RNA in complex with ing/sitting drop vapor diVusion and microbatch. The the U1A RNA binding domain (RBD) protein. This former involves applying 1–10 l of crystallization sample approach dramatically improved the crystallizability of onto a platform (sitting drop) or an inverted glass cover the HDV and hairpin ribozymes [10–12]. The rigid, posi- slide (hanging drop) and allowing it to equilibrate against tively charged U1A protein serves as a structural a much larger volume (0.5 ml or more) of reservoir solu- landmark and forms most of the contacts in the HDV tion through vapor diVusion in a sealed environment. ribozyme crystal lattice, signiWcantly increasing the The latter involves mixing samples with crystallization diVraction limit of the ribozyme crystals [10]. The reagents and allowing the drop to equilibrate with air structure determination process is also expedited by pre- through a layer of oil. Both methods are successful in paring Se-derivatized U1A protein and using multiwave- identifying initial crystallization conditions, while sample length anomalous diVraction (MAD) to solve the phase volumes as small as 20 nl can be set up in automated fash- problem [11,12]. Crystallization modules are usually ion using the microbatch method, enabling high-through- introduced into an RNA molecule through a helical put crystal screening ([1] and references therein). A adaptor segment, whose length can be systematically promising new method using microXuidics, described in varied to allow those modules to sample diVerent orien- this volume of Methods (S. Quake article), enables tations [2]. screening of 48 crystallization conditions, each at three Another successful approach to improving the qual- sample-to-precipitant ratios, with just 3l of sample. ity of RNA crystals has been to use in vitro selection to Despite these approaches, obtaining well-ordered identify more stable folding variants of a native mole- crystals is still seemingly more diYcult for RNA and cule. Crystals of the P4–P6 domain of the Tetrahymena RNA–protein complexes than for proteins. Several fac- group I intron were improved by selection of a P4–P6 tors may contribute to this. Whereas proteins can utilize mutant that folds at a lower magnesium ion concentra- a large number of structural and chemical features on tion than the native sequence [13,14]. To overcome an their molecular surface to form crystal contacts, the apparent Xexibility problem that prevented crystals of repetitive array of negatively charged phosphate groups the Tetrahymena self-splicing group I intron from on the surface of an RNA molecule makes crystal pack- diVracting to high resolution, in vitro selection was used ing more diYcult and potentially error-prone. Hence, to identify active variants of the intron with increased many nucleic acid crystals are poorly ordered and thermal stability [15]. The melting temperature was diVract X-rays to only low resolution [2]. Unlike most increased 10.5 °C in the Wnal selection product, which proteins that fold into globular domains, RNAs fre- contains nine point mutations; most of these seem to quently adopt elongated shapes that pack loosely in improve intramolecular packing interactions in the crystals, with high solvent content. Furthermore, the rel- molecular interior. This variant Tetrahymena intron atively weak tertiary interactions within RNA molecules self-cleaves to a greater extent, although at a slightly lead to more Xexibility and inter-domain movements, slowly rate, than the wild type [15]. Crystals of the Tetra- and a higher tendency to misfold. The former results in hymena intron with a subset of these nine mutations weaker diVraction and higher temperature factors in introduced diVracted to 3.8 Å resolution, a signiWcant RNA crystals, while the latter leads to non-homoge- improvement from 5.0 Å for the wild type crystals (per- neous samples that are hard to crystallize. sonal communication, Dr. Feng Guo, University of Col- Because of these problems, active engineering can be orado at Boulder). very useful for obtaining diVraction quality crystals (for reviews, see [3–5]). A blunt or “sticky” end may be engi- neered into RNA to promote inter-molecular stacking 3. Sample preparation interactions that form “super helices” in the crystal lat- tice [6]. Non-essential regions within the RNA may be Milligram quantities of RNA required for crystalliza- deleted or replaced with stem loops that may potentially tion experiments can be obtained from two sources: form speciWc crystal contacts. For example, GNRA tetr- chemical synthesis and in vitro run-oV transcription aloops and cognate receptor sequences were found to using T7 RNA polymerase. Nucleotide modiWcations mediate crystal contacts in hammerhead ribozyme crys- site-speciWcally incorporated during chemical synthesis tals [7,8]. Engineering GAAA tetraloop–tetraloop recep- can be exploited in structure determination and func- tor interactions into non-essential regions of the tional studies. However, there is an eVective length limit hepatitis delta virus (HDV) ribozyme yielded many of »40 nucleotides for chemical synthesis, as abortive more crystal forms than controls [2]. A similar engineer- products accumulate with each cycle of nucleotide addi- ing approach produced crystals diVracting to 1.5 Å tion. Much larger RNAs can be generated by in vitro 410 A. Ke, J.A. Doudna / Methods 34 (2004) 408–414 transcription reactions. In this case, the RNA coding 75-nucleotide precursor HDV ribozyme was separated sequence is subcloned
Load more

Recommended publications
  • Bottom-Up Self-Assembly Based on DNA Nanotechnology
    nanomaterials Review Bottom-Up Self-Assembly Based on DNA Nanotechnology 1, 1, 1 1 1,2,3, Xuehui Yan y, Shujing Huang y, Yong Wang , Yuanyuan Tang and Ye Tian * 1 College of Engineering and Applied Sciences, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210023, China; [email protected] (X.Y.); [email protected] (S.H.); [email protected] (Y.W.); [email protected] (Y.T.) 2 Shenzhen Research Institute of Nanjing University, Shenzhen 518000, China 3 Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210023, China * Correspondence: [email protected] These authors contributed equally to this work. y Received: 9 September 2020; Accepted: 12 October 2020; Published: 16 October 2020 Abstract: Manipulating materials at the atomic scale is one of the goals of the development of chemistry and materials science, as it provides the possibility to customize material properties; however, it still remains a huge challenge. Using DNA self-assembly, materials can be controlled at the nano scale to achieve atomic- or nano-scaled fabrication. The programmability and addressability of DNA molecules can be applied to realize the self-assembly of materials from the bottom-up, which is called DNA nanotechnology. DNA nanotechnology does not focus on the biological functions of DNA molecules, but combines them into motifs, and then assembles these motifs to form ordered two-dimensional (2D) or three-dimensional (3D) lattices. These lattices can serve as general templates to regulate the assembly of guest materials. In this review, we introduce three typical DNA self-assembly strategies in this field and highlight the significant progress of each.
    [Show full text]
  • Optimal Operation of an Integrated Bioreaction–Crystallization Process
    中国科技论文在线 http://www.paper.edu.cn Chemical Engineering Science 56 (2001) 6165–6170 www.elsevier.com/locate/ces Optimal operation ofan integrated bioreaction–crystallization process for continuous production of calcium gluconate using external loop airlift columns Jie Baoa, Kenichi Koumatsua, Keiji Furumotob, Makoto Yoshimotoa, Kimitoshi Fukunagaa, Katsumi Nakaoa; ∗ aDepartment of Applied Chemistry and Chemical Engineering, Yamaguchi University, Tokiwadai, Ube, Yamaguchi 755-8611, Japan bOshima National College of Maritime Technology, Oshima, Yamaguchi 742-2106, Japan Abstract A kinetic model was proposed to optimize the integrated bioreaction–crystallization process newly developed for production of calcium gluconate crystals using external loop airlift columns. The optimal operating conditions in the bioreactor were determined using an objective function deÿned to maximize the productivity as well as to minimize biocatalyst loss. The optimization of the crystallizer was carried out by matching the crystallization rate to the optimal production rate in the bioreactor because the bioreaction was found to be the rate controlling process. The calcium gluconate productivity under the optimal conditions of the integrated process was obtained by the simulation based on the process model. The productivity ofthe proposed process was found to be comparable to that ofthe current batch fermentationprocess. ? 2001 Elsevier Science Ltd. All rights reserved. Keywords: Process kinetic model; Optimization; Calcium gluconate; Bioreaction–crystallization
    [Show full text]
  • Crystallization in Patterns: a Bio-Inspired Approach**
    PROGRESS REPORTS Crystallization in Patterns: A Bio-Inspired Approach** By Joanna Aizenberg* Nature produces a wide variety of exquisite, highly functional mineralized tissues using simple inorganic salts. Biomineralization occurs within specific microenvironments, and is finely tuned by cells and specialized biomacromolecules. This article surveys bio- inspired approaches to artificial crystallization based on the above concept: that is, the use of organized organic surfaces patterned with specific initiation domains on a sub- micrometer scale to control patterned crystal growth. Specially tailored self-assembled monolayers (SAMs) of x-terminated alkanethiols were micropatterned on metal films using soft lithography and applied as organic templates for the nucleation of calcium carbonate. Crystallization results in the formation of large-area, high-resolution inorganic replicas of the underlying organic patterns. SAMs provide sites for ordered nucleation, and make it possible to control various aspects of the crystallization process, including the precise localization of particles, nucleation density, crystal sizes, crystallographic orientation, morphology, polymorph, stability, and architecture. The ability to construct periodic arrays of uniform oriented single crystals, large single crystals with controlled microporosity, or films presenting patterns of crystals offers a potent methodology to materials engineering. 1. Technological Challenge and Biological Of the many challenges facing materials science, the devel- Inspiration opment of an alternative, bottom±up crystallization route, which would enable the direct, patterned growth of crystals The ability to control crystallization is a critical requirement with controlled physico-chemical properties, became an at- in the synthesis of many technologically important materi- tractive, strategic goal. The fundamental principles of the [12±17] als.[1±5] Crystalline inorganic structures with micrometer-scale bottom±up approach can be borrowed from nature.
    [Show full text]
  • Unsolved Problems in Nanotechnology
    Unsolved Problems in Nanotechnology 61 Biographical sketch of Matthew Tirrell Matthew Tirrell received his undergraduate education in Chemical Engineering at Northwestern University and his Ph.D. in 1977 in Polymer Science from the University of Massachusetts. He is currently Dean of the College of Engineering at the University of California, Santa Barbara. From 1977 to 1999 he was on the faculty of Chemical Engineering and Materials Science at the University of Minnesota, where he served as head of the department from 1995 to 1999. His research has been in polymer surface properties including adsorption, adhesion, surface treatment, friction, lubrication and biocompatibilty. He has co-authored about 250 papers and one book and has supervised about 60 Ph.D. students. Professor Tirrell has been a Sloan and a Guggenheim Fellow, a recipient of the Camille and Henry Dreyfus Teacher-Scholar Award and has received the Allan P. Colburn, Charles Stine and the Professional Progress Awards from AIChE. He was elected to the National Academy of Engineering in 1997, became a Fellow of the American Institute of Medical and Biological Engineers in 1998, was elected Fellow of the American Association for the Advancement of Science in 2000 and was named Institute Lecturer for the American Institute of Chemical Engineers in 2001. 62 Unsolved Problems in Nanotechnology: Chemical Processing by Self-Assembly Matthew Tirrell Departments of Chemical Engineering and Materials Materials Research Laboratory California NanoSystems Institute University of California, Santa Barbara, CA 93106-5130 [email protected] Abstract The many impressive laboratory demonstrations of controllable self-assembly methods generate considerable hope and interest in self-assembly as a manufacturing method for nano-structured products.
    [Show full text]
  • The Bio Revolution: Innovations Transforming and Our Societies, Economies, Lives
    The Bio Revolution: Innovations transforming economies, societies, and our lives economies, societies, our and transforming Innovations Revolution: Bio The The Bio Revolution Innovations transforming economies, societies, and our lives May 2020 McKinsey Global Institute Since its founding in 1990, the McKinsey Global Institute (MGI) has sought to develop a deeper understanding of the evolving global economy. As the business and economics research arm of McKinsey & Company, MGI aims to help leaders in the commercial, public, and social sectors understand trends and forces shaping the global economy. MGI research combines the disciplines of economics and management, employing the analytical tools of economics with the insights of business leaders. Our “micro-to-macro” methodology examines microeconomic industry trends to better understand the broad macroeconomic forces affecting business strategy and public policy. MGI’s in-depth reports have covered more than 20 countries and 30 industries. Current research focuses on six themes: productivity and growth, natural resources, labor markets, the evolution of global financial markets, the economic impact of technology and innovation, and urbanization. Recent reports have assessed the digital economy, the impact of AI and automation on employment, physical climate risk, income inequal ity, the productivity puzzle, the economic benefits of tackling gender inequality, a new era of global competition, Chinese innovation, and digital and financial globalization. MGI is led by three McKinsey & Company senior partners: co-chairs James Manyika and Sven Smit, and director Jonathan Woetzel. Michael Chui, Susan Lund, Anu Madgavkar, Jan Mischke, Sree Ramaswamy, Jaana Remes, Jeongmin Seong, and Tilman Tacke are MGI partners, and Mekala Krishnan is an MGI senior fellow.
    [Show full text]
  • Important Factors Influencing Protein Crystallization
    Global Journal of Biotechnology and Biomaterial Science Mohnad Abdalla1*, Wafa Ali Eltayb1, Mini Review Abdus Samad1, Elshareef SHM1 and TIM Dafaalla2 1Hefei National Laboratory for Physical Sciences at the Important Factors Influencing Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Protein Crystallization Republic of China 2College of Plant Science, Jilin University, Changchun 130062, China Abstract Dates: Received: 16 September, 2016; Accepted: The solution of crystallization problem was introduced around twenty years ago, with the 29 September, 2016; Published: 30 September, introduction of crystallization screening methods. Here reported some of the factors which affect 2016 protein crystallization, solubility, Concentration of precipitant, concentration of macromolecule, ionic *Corresponding author: Mohnad Abdalla, strength, pH, temperature, and organism source of macromolecules, reducing or oxidizing environment, Hefei National Laboratory for Physical Sciences additives, ligands, presence of substrates, inhibitors, coenzymes, metal ions and rate of equilibration. at the Microscale and School of Life Sciences, The aim of this paper to give very helpful advice for crystallization. University of Science and Technology of China, Hefei, Anhui 230027, PR China, E-mail: Buffer is most straight forward way to make a crystallization www.peertechz.com problem by effect the protein behave. There are many rules and Keywords: Proteins; Crystallization; Isoelectric point; different protein crystallization methods, some of it has been pH; Solubility; Stability developed during the recent years. However, Protein crystallization still represents a great challenge for bio crystallography. The Introduction cumulation of crystallization information in crystallization databases and in structural articles it allow us to design crystallization X-ray crystallography has provided 3D structures of thousands experiments depending on the character of the protein.
    [Show full text]
  • Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology
    Journal of Visualized Experiments www.jove.com Video Article Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology Fabrice Gorrec1, Jan Löwe1 1 Laboratory of Molecular Biology, Medical Research Council Correspondence to: Fabrice Gorrec at [email protected] URL: https://www.jove.com/video/55790 DOI: doi:10.3791/55790 Keywords: Biochemistry, Issue 131, X-ray crystallography, protein crystallization, high-throughput screening, fully automated system, nanoliter droplets, vapor diffusion, initial screen, nanoliter dispenser, liquid handler, crystal optimization, 4-corner method, additive screening Date Published: 1/24/2018 Citation: Gorrec, F., Löwe, J. Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology. J. Vis. Exp. (131), e55790, doi:10.3791/55790 (2018). Abstract When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.
    [Show full text]
  • A Guide to the Crystallographic Analysis of Icosahedral Viruses
    UC Irvine UC Irvine Previously Published Works Title A guide to the crystallographic analysis of icosahedral viruses Permalink https://escholarship.org/uc/item/64w5j3pz Journal Crystallography Reviews, 21(1-2) ISSN 0889-311X Authors McPherson, A Larson, SB Publication Date 2015 DOI 10.1080/0889311X.2014.963572 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Crystallography Reviews, 2015 Vol. 21, Nos. 1–2, 3–56, http://dx.doi.org/10.1080/0889311X.2014.963572 REVIEW A guide to the crystallographic analysis of icosahedral viruses Alexander McPherson∗ and Steven B. Larson Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA (Received 13 August 2014; accepted 5 September 2014) Determining the structure of an icosahedral virus crystal by X-ray diffraction follows very much the same course as conventional protein crystallography. The major differences arise from the relatively large sizes of the particles, which significantly affect the data collection process, data processing and management, and later, the refinement of a model. Most of the other differences are due to the high 532point group symmetry of icosahedral viruses. This alters dramatically the means by which initial phases are obtained by molecular substitu- tion, extended to higher resolution by electron density averaging and density modification, and the refinement of the structure in the light of high non-crystallographic symmetry. In this review, we attempt to lead the investigator through the various steps involved in solving the structure of a virus crystal. These steps include the purification of viruses, their crystal- lization, the recording of X-ray diffraction data, and its reduction to structure amplitudes.
    [Show full text]
  • Crystallization and Structure Analysis
    Electronic Supplementary Material (ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2013 1 ELECTRONIC SUPPLEMENTARY INFORMATION Table of Contents 1. Experimental procedures 1 Production and purification of sermetstatin 1 Production and purification of snapalysin 2 In vitro activation studies of prosnapalysin 3 Proteolytic and inhibition assays 4 Cleavage of sermetstatin mutants 4 Complex formation and purification 5 Crystallization and X-ray diffraction data collection 5 Structure solution and refinement 6 Miscellaneous 8 2. Acknowledgments 8 3. Supplemental References 9 4. Supplemental Tables 11 Supplemental Table S1 11 Supplemental Table S2 12 Supplemental Table S3 13 5. Supplemental Figures 14 Supplemental Figure S1 14 1. Experimental procedures Production and purification of sermetstatin – A synthetic gene coding for sermetstatin from Streptomyces caespitosus (UniProt database code Q9FDS0), also known as Streptomyces caespitosus neutral proteinase inhibitor 1, was purchased (GenScript) and cloned into a modified pET-32a vector between the BglII and HindIII restriction sites. This vector attaches an N-terminal thioredoxin-His6 fusion construct followed by a tobacco-etch-virus (TEV) protease recognition site. Sermetstatin was produced by heterologous overexpression in Escherichia coli Origami2 (DE3) cells (Novagen). These were grown at 37°C in Luria-Bertani (LB) medium containing 100µg/ml ampicillin and 10µg/ml tetracycline, induced at an OD550 of 0.6 with isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.25mM, and subsequently incubated overnight at 18°C. Cultures were centrifuged at 7,000xg for 30min at 4°C. Pellets were washed twice with buffer A (50mM Tris-HCl, 500mM NaCl, pH8.0) and resuspended in the same buffer further containing 20mM imidazole and supplemented with EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics) and DNase I (Roche Diagnostics).
    [Show full text]
  • 8.6 - Two-Solvent Recrystallization Guide
    8.6 - Two-Solvent Recrystallization Guide Overview: For a two-solvent recrystallization, you should have one solvent (solvent #1) in which your desired compound is soluble at the boiling point. The second solvent (solvent #2) should induce crystallization when added to a saturated solution of your compound in the primary solvent. Reference: See Zubrick pages 105-106, Mohrig 183-197 Recrystallization Steps: 1) The first step is to remove insoluble material from your compound by filtration. 2) Transfer the material to a 50-mL Erlenmeyer flask, equipped with a stir bar. Add an excess amount of solvent #1 (about 20 mL in this case) and heat to boiling on a stir/hot plate. The excess solvent is used to keep your compound from precipitating during the filtration. 3) Filter off the insoluble contaminant through fluted filter paper in a pre-warmed stemless funnel (pre-warm by adding some hot solvent just before you filter your solution, thus preventing loss of material on the filter paper.) 4) Wash the flask and filter paper with about 2 mL of hot solvent. 5) Reduce the volume of your solution to about 15 mL by boiling off the excess solvent. 6) Cool to room temperature. At this point, you probably do not have a saturated solution, so crystallization will not occur. 7) Add solvent #2 dropwise until the solution just becomes cloudy. Again heat the solution to the boiling point (with stirring!) and continue the addition of solvent #2. After each drop, you will notice a cloudiness that dissolves away. Continue dropwise addition 66 of solvent #2 until the solution is saturated (i.e.
    [Show full text]
  • Precipitation and Crystallization Processes
    Precipitation and Crystallization Processes Gordon Jarvenin Los Alamos National Laboratory Introduction Precipitation and crystallization refer to unit operations that generate a solid from a supersaturated solution. The non-equilibrium supersaturated condition can be induced in a variety of ways such as removal of solvent by evaporation, addition of another solvent, changes of temperature or pressure, addition of other solutes, oxidation-reduction reactions, or even combinations of these. The distinction between precipitation and crystallization is quite often based on the speed of the process and the size of the solid particles produced. The term precipitation commonly refers to a process which results in rapid solid formation that can give small crystals that may not appear crystalline to the eye, but still may give very distinct x-ray diffraction peaks. Amorphous solids (at least as indicated by x-ray diffraction) may also be produced. The term precipitation also tends to be applied to a relatively irreversible reaction between an added reagent and other species in solution whereas crystallization products can usually be redissolved using simple means such as heating or dilution. Precipitation processes usually begin at high supersaturation where rapid nucleation and growth of solid phases occur. In both precipitation and crystallization processes the same basic steps occur: supersaturation, nucleation and growth. Nucleation does not necessarily begin immediately on reaching a supersaturated condition, except at very high supersaturation, and there may be an induction period before detection of the first crystals or solid particles. Nucleation can occur by both homogeneous and heterogeneous processes. In general, homogeneous nucleation is difficult to achieve because of the presence of heteronuclei from colloids, dust, or other foreign material in the solution.
    [Show full text]
  • The Roots—A Short History of Industrial Microbiology and Biotechnology
    Appl Microbiol Biotechnol (2013) 97:3747–3762 DOI 10.1007/s00253-013-4768-2 MINI-REVIEW The roots—a short history of industrial microbiology and biotechnology Klaus Buchholz & John Collins Received: 20 December 2012 /Revised: 8 February 2013 /Accepted: 9 February 2013 /Published online: 17 March 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Early biotechnology (BT) had its roots in fasci- mainly secondary metabolites, e.g. steroids obtained by nating discoveries, such as yeast as living matter being biotransformation. By the mid-twentieth century, biotech- responsible for the fermentation of beer and wine. Serious nology was becoming an accepted specialty with courses controversies arose between vitalists and chemists, resulting being established in the life sciences departments of several in the reversal of theories and paradigms, but prompting universities. Starting in the 1970s and 1980s, BT gained the continuing research and progress. Pasteur’s work led to the attention of governmental agencies in Germany, the UK, establishment of the science of microbiology by developing Japan, the USA, and others as a field of innovative potential pure monoculture in sterile medium, and together with the and economic growth, leading to expansion of the field. work of Robert Koch to the recognition that a single path- Basic research in Biochemistry and Molecular Biology dra- ogenic organism is the causative agent for a particular matically widened the field of life sciences and at the same disease. Pasteur also achieved innovations for industrial time unified them considerably by the study of genes and processes of high economic relevance, including beer, wine their relatedness throughout the evolutionary process.
    [Show full text]