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

419 Pages·2014·6.39 MB·English

by  W. T. Godbey

An Introduction to Biotechnology
An Introduction
to Biotechnology
The science, technology and medical
applications
W. T. Godbey
Tulane University
New Orleans, Louisiana
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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 P artial 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