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An Introduction to Biotechnology: The Science, Technology and Medical Applications PDF

419 Pages·2014·41.347 MB·English
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An Introduction to Biotechnology An Introduction to Biotechnology The science, technology and medical applications W. T. Godbey Tulane University New Orleans, Louisiana AMSTERDAM (cid:127) BOSTON (cid:127) HEIDELBERG (cid:127) LONDON NEW YORK (cid:127) OXFORD (cid:127) PARIS (cid:127) SAN DIEGO SAN FRANCISCO (cid:127) SINGAPORE (cid:127) SYDNEY (cid:127) TOKYO Academic Press is an imprint of Elsevier Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright © W T Godbey, 2014. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notice Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-1-907568-28-2 For information on all Academic Press publications visit our website at elsevierdirect.com Typeset by SPi Global, India Printed and bound in China 14 15 16 17 10 9 8 7 6 5 4 3 2 1 List of Figures Figure 1.1 The eukaryotic plasma membrane 2 Figure 1.2 The structure of glycerol 2 Figure 1.3 The structure of phosphatidic acid 3 Figure 1.4 Common phospholipids found in the plasma membrane 3 Figure 1.5 The structure of sphingomyelin 4 Figure 1.6 Distribution of membrane phospholipids 5 Figure 1.7 The structure of cholesterol 6 Figure 1.8 Cholesterol fits between adjacent phospholipids 6 Figure 2.1 General structure of an amino acid 10 Figure 2.2 l- and d-forms of the general amino acid structure 10 Figure 2.3 structures of the 20 common amino acids 11 Figure 2.4 Ionization states of glycine 14 Figure 2.5 Ionization states of lysine 15 Figure 2.6 Amino acid polymerization reaction 16 Figure 2.7 Amino acid placement and interactions in the alpha helix 17 Figure 2.8 Beta pleated sheets 18 Figure 2.9 Beta turn 19 Figure 2.10 Proline in cis and trans conformations 20 Figure 2.11 the hydrophobic effect 21 Figure 2.12 A protein active site 22 Figure 2.13 Transmembrane proteins 23 Figure 2.14 Membrane protein migration 25 Figure 2.15 Barriers to membrane protein migration 27 Figure 2.16 Structures: soap versus detergent 28 Figure 2.17 General structure of a surfactant and a micelle 29 Figure 2.18 The structure of Triton X-100 30 Figure 3.1 Relative membrane permeabilities 36 Figure 3.2 Symport, antiport, active and passive transport 36 Figure 3.3 The mechanism of the sodium/glucose symporter 38 Figure 3.4 Examples of transporters that control cytosolic pH 40 Figure 3.5 Actions of two proton ATPases 41 Figure 3.6 A cell with labeled lysosomes 42 Figure 3.7 The sodium/potassium ATPase 43 Figure 3.8 The sodium-driven calcium exchanger, and an active Ca2+ transporter 44 Figure 3.9 IgG structure 48 Figure 3.10 Phagocytosis 50 Figure 3.11 A triskelion 52 Figure 3.12 Clathrin-coated pits 53 Figure 3.13 Structure of an LDL complex 56 Figure 3.14 Receptor recycling: the LDL receptor 57 Figure 3.15 Reasons for poor cholesterol uptake 58 Figure 3.16 Receptor recycling: the transferrin receptor 59 ix x List of Figures Figure 3.17 Transcytosis 61 Figure 4.1 The structures of ribose, phosphoribose, and an RNA dinucleotide 66 Figure 4.2 Structures of NMP, dNMP, and NTP 68 Figure 4.3 RNA polymerization 69 Figure 4.4 Structures of the most common nucleotide bases 70 Figure 4.5 The structure of the trinucleotide CTG 72 Figure 4.6 Summary of the Hershey-Chase experiments 73 Figure 4.7 DNA base pairing 74 Figure 4.8 dsDNA melting temperatures 75 Figure 4.9 Summary of the Meselson-Stahl experiments 76 Figure 4.10 The genetic code 79 Figure 4.11 Schematic of a tRNA molecule and its anticodon 80 Figure 4.12 Schematic of the ribosome with E, P, and A sites 80 Figure 4.13 Schematic of ribosomal interactions with tRNA during translation 81 Figure 4.14 Logical components of a gene 83 Figure 4.15 The number of genes in the human genome 85 Figure 4.16 Structures of the H antigen 86 Figure 4.17 Blood types 87 Figure 4.18 Structure of the 5’ RNA cap 91 Figure 4.19 Steps in the creation of the 5’ RNA cap 92 Figure 4.20 Splicing, or the removal of introns from eukaryotic RNA 93 Figure 4.21 The eukaryotic poly(A) tail 94 Figure 4.22 The eukaryotic transcription machinery 99 Figure 4 Aside: Ether, ester, and phosphodiester structures 67 Figure 5.1 Schematic of the cell cycle (S, M, and G phases) 108 Figure 5.2 Schematic of the cell cycle (IPMAT phases) 108 Figure 5.3 Chromosomal locations during mitosis 109 Figure 5.4 Centromere, kinetochore, kinteochore microtubules, and mitotic spindle 110 Figure 5.5 Cell cycle checkpoints 111 Figure 5.6 Quantitation of DNA concentration during the cell cycle 113 Figure 5.7 A growth curve 115 Figure 5.8 Growth curves to consider 118 Figure 5.9 Cryptic growth 119 Figure 5.10 Diauxic growth 120 Figure 5.11 Growth curves for different carbon-source affinities 123 Figure 5.12 A hemacytometer 126 Figure 5.13 A hemacytometer with cells for counting 127 Figure 5.14 Schematic of an agar plate with colonies 129 Figure 5.15 Schematic of a cell counter 131 Figure 5.16 A spinner flask 135 Figure 6.1 Sketch of Gram-positive and Gram-negative bacteria 144 Figure 6.2 The structure of crystal violet 145 Figure 6.3 Crystal violet without and with a mordant 145 Figure 6.4 Gram stain results 146 Figure 6.5 A fly trapped in amber 148 Figure 6.6 Sterile vs. disinfected vs. sanitized 149 Figure 6.7 The structure of ethylene oxide 150 Figure 6.8 A sterilized liquid for drinking? 150 Figure 6.9 The structures of several common alcohols 154 Figure 6.10 Potential targets for antimicrobial drugs 155 Figure 6.11 NAM and NAG structures 156 List of Figures xi Figure 6.12 NAM and NAG crosslinking in the prokaryotic cell wall 157 Figure 6.13 How folate is used in purine synthesis 159 Figure 6.14 The structures of PABA, sulfa drugs, and folic acid 160 Figure 6.15 The structure of polymyxin B 161 Figure 7.1 An RGD sequence 166 Figure 7.2 Okazaki fragments 168 Figure 7.3 Telomeres, as detected by FISH 169 Figure 7.4 Normal vs. cancer cells 171 Figure 8.1 Illustration of one of Stokes’ experiments 174 Figure 8.2 Illustration of one of Stokes’ experiments 174 Figure 8.3 Illustration of one of Stokes’ experiments 175 Figure 8.4 Illustration of one of Stokes’ experiments 176 Figure 8.5 The Jablonski diagram used to explain fluorescence 178 Figure 8.6 The structure of quinine 179 Figure 8.7 A hypothetical fluorescence curve 180 Figure 8.8 (Slightly more than) the visible light spectrum with wavelengths 182 Figure 8.9 Fluorescence spectra overlain to demonstrate FRET 184 Figure 9.1 Overview of the deletion analysis process 188 Figure 9.2 A straightforward way to map out deletion analysis data 189 Figure 10.1 The structure of agarose 194 Figure 10.2 The structure of EDTA 194 Figure 10.3 Removing the comb from a cast agarose gel 194 Figure 10.4 The structure of ethidium bromide 195 Figure 10.5 Ethidium bromide used to visualize DNA via a UV-light box 196 Figure 10.6 DNA footprinting 199 Figure 10.7 Interpreting DNA footprinting data 200 Figure 11.1 The first two cycles of a hypothetical PCR set 209 Figure 11.2 The third cycle of a hypothetical PCR set 210 Figure 11.3 The number of copies of certain dsDNA species generated by PCR 211 Figure 11.4 PCR amplification curve 214 Figure 11.5 cDNA doublings amidst noise 216 Figure 11.6 The problem with running traditional PCR for too many cycles 219 Figure 11.7 Common problems encountered with traditional PCR 220 Figure 11.8 Two sources of primer dimers 221 Figure 11.9 Why SYBR green can be used to monitor PCR progress 223 Figure 11.10 Real-time PCR fluorescence curve 224 Figure 11.11 qPCR output using an internal control 226 Figure 11.12 The threshold value chosen in qPCR is somewhat arbitrary 229 Figure 11.13 Error magnitude versus cycle number 229 Figure 11.14 Mechanism behind primer/probe sets 232 Figure 12.1 Map of the plasmid pEGFP-N1 238 Figure 12.2 Partial restriction map of pBmp5, a fictitious plasmid used as an example 240 Figure 12.3 Plasmid pBMP5 cut open 241 Figure 12.4 Gel of Plasmid pBMP5 cut open 242 Figure 12.5 Partial restriction map of pBmp5-b, another fictitious plasmid used as an example 246 Figure 12.6 Blunting sticky ends with the Klenow fragment 246 Figure 12.7 Detection of insert in reverse orientation 247 Figure 12.8 Mechanism of DNA ligase 248 Figure 12.9 Alkaline phosphatase prevents self-ligation 249 Figure 12.10 Adding a restriction site to an amplicon 251 Figure 12.11 LB agar with bacterial colonies growing 253 xii List of Figures Figure 12.12 Logical progression to deciding which colony is appropriate for further amplification 256 Figure 12.13 The problem of double-ligations 257 Figure 12.14 Detecting multiple sticky inserts 258 Figure 12.15 Location of DNA in the bacterial cell 261 Figure 12.16 Absorption spectrum for dsDNA 265 Figure 12.17 Determining DNA purity 269 Figure 12 Aside figure Osmolarity 259 Figure 13.1 Organization of some common gene delivery methods/vehicles 276 Figure 13.2 Basic structure of a retrovirus 278 Figure 13.3 The basic structure of an adenovirus 281 Figure 13.4 A gene gun 286 Figure 13.5 Microinjection of a frog oocyte 288 Figure 13.6 Polymerization of branched PEI 291 Figure 13.7 Divergent or convergent pathways of dendrimer construction 293 Figure 13.8 G3 and G4 dendrimers 294 Figure 13.9 The structures of chitin and chitosan 297 Figure 13.10 Structures predicted by the packing parameter P 299 Figure 13.11 The structure of DOTMA 300 Figure 13.12 The structure of DOTAP 300 Figure 13.13 The structure of DC-Chol 300 Figure 13.14 The structure of DOSPA 301 Figure 13.15 The structure of DOGS 301 Figure 13.16 The structure of DOPE (top) and DOPC (bottom) 302 Figure 13.17 PEI structure 305 Figure 13.18 The structure of the basic repeating unit of PLL 305 Figure 14.1 Petunias from the Jorgensen experiments 315 Figure 14.2 Schematic of the actions of Dicer and RISC 318 Figure 14.3 Pri- and pre-miRNA 320 Figure 15.1 DNA fingerprinting example using RFLPs 324 Figure 15.2 DNA fingerprinting – crime scene example 326 Figure 15.3 Names and relative positions of the 13 core CODIS STR loci on human chromosomes 328 Figure 16.1 The Embden-Meyerhof pathway of glycolysis 332 Figure 16.2 The Entner-Douderoff pathway of glycolysis 336 Figure 16.3 Fermentation of glucose into two lactic acid molecules 337 Figure 16.4 Common fermentation products 339 Figure 16.5 Malted barley 340 Figure 16.6 Mashing 341 Figure 16.7 Mature mash 341 Figure 16.8 Dried leaf hops 342 Figure 16.9 A mature yeast starter culture 343 Figure 16.10 News articles on food riots 347 Figure 16.11 Centrifugal pump 348 Figure 16.12 Poly(glucose), alpha- and beta-linkages 350 Figure 17.1 A morula 354 Figure 17.2 A blastocyst 354 Figure 17.3 Gastrula cross section with germ cell lineages 355 Figure 17.4 Levels of cellular potential and related cell types 356 Figure 17.5 Ear on a mouse back 359 Figure 17.6 Tissue engineered artery 360 Figure 17.7 Tissue culture dishes and flasks in an incubator 362 Figure 17.8 Many sizes of tissue culture plates and flasks 364 List of Figures xiii Figure 17.9 Multilayered tissue culture flask 364 Figure 17.10 Spinner flask 365 Figure 17.11 Scheme for a 2-D perfusion bioreactor 366 Figure 17.12 A 3-D perfusion bioreactor set up in an incubator 366 Figure 17.13 Muscle cells line up upon exposure to shear 367 Figure 17.14 Glycolic acid is polymerized to form poly(glycolic acid) 367 Figure 17.15 Lactic acid is polymerized to form poly(lactic acid) 368 Figure 17.16 Cylindrical PLA scaffolds 368 Figure 17.17 Examples of copolymer architectures 369 Figure 17.18 Structure of an artery 370 Figure 18.1 Frost on berries (ice-minus bacteria can inhibit this) 376 Figure 18.2 Non-Bt versus Bt plant 376 Figure 18.3 The structure of glyphosate 377 Figure 18.4 Roundup Ready™ soybeans 378 Figure 18.5 Not all dirt is the same 380 Figure 18.6 Cans of puree from genetically modified tomatoes 382 Figure 18.7 IR8 rice field comparison 385 Figure 18.8 Farmers receive IR8-288-3 386 Figure 18.9 The cleavage of beta-carotene yields two molecules of vitamin A 387 Figure 18.10 Structure of a rice grain 388 Figure 18.11 Engineered biochemical pathway to get from geranylgeranyl diphosphate to beta-carotene, used by golden rice 388 Figure 19.1 Intellectual Property Offices around the world 394 Figure 19.2 Chemical structures of minoxidil (Rogaine) and atorvastatin (Lipitor) 395 Figure 19.3 A sample nondisclosure agreement 398 List of Tables Table 4.1 Names of Purine and Pyrimidine Bases, Nucleosides, and Nucleotides 71 Table 4.2 The Consensus Sequence of the TATA Box in Yeast, Worked Out From All Possible Eight-Mers of [T A (A/T) (A/T) N N N N] 95 Table 4.3 The Consensus Sequences of Several Transcriptional Regulatory Elements 97 Table 4.4 Hypothetical Plasmids and the Amount of Gene Expression Attainable with various Gene Elements 100 Table 4.5 A Shortened List of Initiation Factors and Their Roles in Ribosomal Assembly 102 Table 6.1 General Resistance of Contaminants to Physical and Chemical Methods of Control 146 Table 10.1 Size Ranges of dsDNA Fragments That can be Resolved Using the Given Percentages of Standard Agarose 195 Table 16.1 Energy Considerations for Butanol versus Ethanol 349 xv Preface This book was written for the student with little to no biology background. The goal of the book is to introduce the student to the world of biotechnology but in a way that runs deeper than a mere survey. There are a myriad of biotechnolo- gies in the world today, and the number continues to grow. I happen to find the world of biotechnology a very exciting world, and I want to share that excite- ment with you, the reader. However, to fully appreciate just how cool some of these technologies are, one must understand some of the science underlying the glamour. This is not intended to be a comprehensive book on all biotechnologies. One could spend the better part of a decade becoming an expert in only one area. Likewise, while I state that one must understand the science behind a technol- ogy to fully appreciate it—and healthy portion of that science is covered in this book—the text is not intended to be a complete, rigorous reference book for the biological or chemical sciences. Instead, it was my aim to produce a book that serves as a mix of both basic science and biotechnological applications so that you, the reader, might become energized about part or all of the field on a level that is deep enough to allow you to continue further pursuits of it with the knowl- edge that a solid foundation has been laid. If you still have a passion for one of the subjects contained in this book after studying it, that passion is probably quite real. I want you to get enough foundation so that you can appreciate what is out there in the world of biotechnology, so that you will understand new develop- ments in greater depth than what you might hear in the news, and so that you will not be fooled by unsubstantiated claims you might read on the Internet. The book is divided into three units. In the first, basic science is covered to introduce the reader to the cell, how it behaves, and what it is made of. For instance, if you want to design a drug that you want to enter the cell, you must know and understand the barrier that separates the inside of the cell from the exterior environment. If you want that drug to affect how the cell is behaving, then you should understand how a cell functions so that you can pick a cellular target upon which your drug can act. Perhaps, you want the cell to produce a product that you can isolate and sell, such as recombinant insulin, ethanol, or a novel protein that you have designed. To be successful in this endeavor, you must understand how the cell would go about producing the product, in addi- tion to knowing exactly what your product would be. While we do not cover every possible product that could be produced by a cell, we do cover some of the building blocks such as amino acids/proteins and nucleic acids/DNA/RNA. xvii Godbey, 978-1-907568-28-2

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