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BIOTECHNOLOGY
MODERN
BIOTECHNOLOGY
Defining and Solving
HumanProblems
Abstract
Biotechnology is a fascinating interdisciplinary field uniquely poised to
take on some of the worlds most complex problems. With this thesis at
its core, Modern Biotechnology: Defining and Solving Human Problems
takes a refreshing problems-based approach to exploring the field. Novice
readers will come away with a broad appreciation for the significance
of current and emerging biotechnologiesfrom regenerative medicine,
to genetically enhanced crops, to biofuels. Experts will benefit from
the concise review of timely game-changing technologies such as DNA
sequence-by-synthesis and genome editing. Despite being set within a
conceptual framework of wicked problems (i.e., disease, food production, environmental degradation), insights into the current state and future
potential of biotechnologies make this book both optimistic and forward
thinking. This is not just an informative textits an entry point into a
discipline with the potential to change the world.
KEYWORDS
bioassay, biofuel, biopharmaceuticals, bioremediation, bioreporters, biosensors, biotechnology, cancer, cloning, CRISPR/Cas9, DNA sequencing, drug development, gene therapies, genetically modified crops, HIV,
interdisciplinary, molecular diagnostic, omic technologies, recombinant
DNA, stem cells, systems thinking, transgenic animals, vaccines, wicked
problems
Contents
List of Figures
xi
List of Tables
xiii
List of Boxes
xv
Acknowledgments
xvii
1 Introduction
1
1.1 What is Biotechnology?
1
1.2Milestones in the Development of Modern Biotechnology 4
1.3 Wicked Problems
14
1.4 Brief Summary
16
2 Biological Foundations
2.1 Biological Parts
2.2 Modern Tools of the Trade
2.3 Brief Summary
17
17
30
54
3 Disease
3.1 The Problem: Disease
3.2 Better Medical Interventions
3.3 Brief Summary
55
55
62
85
4 Food Production
4.1 The Problem: Food Production
4.2 Safer Foods
4.3 Better Crops and Livestock
4.4 Brief Summary
87
87
89
93
99
5 Environmental Degradation
5.1 The Problem: Pollution
5.2 Better Environmental Monitoring
5.3 Better Environmental Clean-Up
101
101
102
104
x Contents
107
111
6 Conclusions
6.1 Defining and Solving Problems
6.2 Actualizing Biotechnologys Potential
6.3 Brief Summary
113
113
115
118
Notes
119
References
131
Index
149
List of Figures
Figure 1.1. Forms of organismal cloning.
19
20
28
32
33
38
40
45
47
49
61
61
69
77
84
91
96
104
List of Tables
Table 1.1. General categories of biotechnologies
27
64
94
114
List of Boxes
Box 1.1. Misconceptions about organismal cloning
11
14
32
38
59
65
70
71
74
75
79
81
83
84
88
93
97
99
102
103
107
109
110
117
Acknowledgments
I thank Kristine and Dennis Batchelet and Kris Hensley for their unique
support and encouragement during the preparation of this book. Brooke
Jude, PhD inspired and cheered me on. I thank my friend and colleague,
Ron Raab, PhD, for his time and expertise in the careful reading of my
manuscript. My husband, Michael Stockwell, played a pivotal and essential role throughout the project. I sincerely thank him for his encouraging words, p robing questions, challenging insights, and ever-thoughtful
suggestions.
CHAPTER 1
Introduction
1.1 WHAT IS BIOTECHNOLOGY?
Biotechnology is the application of biologynot the basic science of
biological phenomena. Where a biologist asks, How does this biological
system work? a biotechnologist asks, How can I use it? More precisely,
biotechnology is the development of tools that aim to define or solve
human problems. They are inspired by biological processes and are made
of biological components. For example, a molecular diagnostic defines
diseased versus healthy states. A vaccine given to livestock can curb the
spread of foodborne disease. A genetically engineered microbe can clean
up an oil spill. More generally, biotechnologies most often belong to one
of five primary categories, as described in Table 1.1.
When defining biotechnology, its helpful to identify what biotechnology is not. Biotechnology is not biomedical engineering. Biotechnologies do not include medical imaging systems such as X-ray and magnetic
resonance imaging (MRI), or prosthetic devices such as artificial limbs.
These examples are not biotechnologiesalthough they interface with
biological systemsbecause they do not entail a transformation by a
biological process and are not made of biologically derived components.
1.1.1BIOTECHNOLOGY IS INHERENTLY
INTERDISCIPLINARY
Modern biotechnology emerged in the early 1970s and originally involved
the biomanufacture of cellular or viral componentsthat is molecular
parts and machinesto perform specific tasks. Since genetic engineering
is central to this work, and unicellular microbes are highly genetically
amenable, the study of microscopic organisms and their genes (i.e., microbial genetics) is a critical component of biotech. Biotechnologists most
2 MODERN BIOTECHNOLOGY
Genetically modified
organisms (GMOs)
General purpose
Study the components and interactions
within biological systems
Measure molecular imbalances within
biological or ecological systems
Train the immune system to fight disease
Replace or supplement biological s ystem
components; sold as commercial
products
Perform desirable biomanufacturing/
biotransformation activities; sold as
enhanced commercial products
commonly employ the tools of viruses, bacteria, and yeast. The development of new biofuels is making microalgae cultivation significant as well.
Viruses supply the coding sequence for unique protein machines, such
as reverse transcriptase,1 which do not typically exist in cells. Bacteria
serve as genetically tractable and easily propagated cellular factories for
the biomanufacture of biological products such as proteins and other small
molecules. Yeast cells are similarly easy to grow and manipulate. They are
best-suited for manufacturing biological components derived from higher
organisms (e.g., humans) because they are relatively adept at performing
the structural modifications typical of more complex organisms.
Biotechnology is also part biochemistry, engineering, and computer
science. Biochemistry is applied to help understand the dynamics of biological systemshow the parts fit and work together to achieve observable
emergent properties. As design and fabrication are critical components of
product development, biotechnologists must employ principles from the
engineers tool kitabstraction, standardized parts, and iterative design
(i.e., cycles of design/build/test). Other invaluable toolsmodeling and
the mining of large data setsare supported by computation.
It may seem that the flow of information goes in one directionfrom
the basic scientists (e.g., biologists, chemists) to the biotechnologists. This
is not always the case. Much is learned about the underlying principles of a
system through its construction and analysis. The physicist, Richard Feynman famously wrote, What I cannot create, I do not understand. This quote
has been the calling card of many synthetic biologists,2 belonging to a specialized branch of biotechnology in which biological parts are mixed-andmatched to create largely new biological systems. This is achieved through
Introduction3
computer-aided design of complex systems followed by de novo (i.e., created from scratch) deoxyribonucleic acid (DNA) synthesis and assembly.
1.1.2 BIOTECHNOLOGY IS MISUNDERSTOOD
Biotechnology is a far-reaching fieldone that overlaps with and is bound
by the limits of science, technology, ethics, policy, and regulation. Thus,
stakeholders exist in the communities of academia, industry, government,
and consumers. Despite its significant impact on and intersection with
other mainstream natural sciences, biotechnology is largely misunderstood. You may be hard-pressed to find scientists who identify as biotechnologists, although many practice it. Why is this? Most likely its the
result of the interdisciplinary nature of biotech and the relative rarity of
undergraduate programs that don the biotechnology name. There are just
182 Biotechnology degree-conferring schools in the United States, with
the following breakdown: 63 Associates, 91 Bachelors, 58 Masters, and
10 PhD programs (Guide 20072014). Those who practice biotechnology
are more likely to identify as geneticists or micro-, cell, molecular, or even
computational biologists. These are the names of the more traditional programs of study from which they hold degrees.
Other biotechnology stakeholders may not appreciate the role they
play in the system. For example, just as an applied microbiologist may not
identify as a biotechnologist, a patient undergoing a blood test at a clinic
may not identify as a consumer, and thus supporter of b iotechnology.
A humanitarian donating money to a charity may not realize that his/
her funds could be used to purchase biotechnologies such as vaccines,
biopharmaceuticals, organisms genetically modified (GM) to improve
crop yields or to clean up environmental contaminants. A policy-maker
who votes in favor of universal health care may not appreciate his/her
indirect support of medical biotechnologies such as regenerative and
personalized medicines.
With misunderstanding comes under-appreciation, if not fear. The
name biotechnology may be off-puttingbeing a combination of bio
(that which is natural, and perhaps good) and technology (that which
is unnatural, manmade, and manipulated, and perhaps not good). This
is the fuel for science fiction turned ill-advised biotechnology phobias.
Visions of frankenfoods, disease-causing vaccines,3 or hidden facilities
housing organ-donating human clones may come to mind. Even high
power policy-makers fall victim to the images invented by science fiction.
In 2004, a member of the Presidents Council on Bioethics referred to the
sci-fi movie GATTACA4 as if it was real, in an article he wrote in The
4 MODERN BIOTECHNOLOGY
Atlantic (Guyer and Moreno 2004; Sandel 2004). This example is not an
exception to the rule (Guyer and Moreno 2004).
Thus, when exploring the field of biotechnology, one must be
mindful of both its scientific and the ethical limitations. Like any technology, it is subject to one great shortcomingthe ability to be used in
an inappropriate or harmful manner. It is the discretion of the user, not the
technology itself, which is in question. Thus, those working in the field
of biotechnology must strive to maintain transparency within the system,
such that misunderstandings can be rectified and the appropriate checks
and balances among the stakeholders be upheld.
Introduction5
6 MODERN BIOTECHNOLOGY
Introduction7
8 MODERN BIOTECHNOLOGY
into a final version (Green, Watson, and Collins 2015; Institute 2016b).
All told, thanks to the unprecedented collaboration between thousands of
international scientists, the HGP took just 13 years and ~$2.7billion13 to
complete (Institute 2016b). Some 25 years after this grand project began,
researchers are still assigning meaning to the sequences obtained (Green,
Watson, and Collins 2015). Being the first large-scale project of its kind,
the HGP modeled a new kind of researchconsortium-based and interdisciplinarywhat some are calling big science (Green, Watson, and
Collins 2015).
As the HGP unfolded, sequencing and computational technologies
were invented and modified to meet the needs of the project. For example,
whole genome sequencing of less complicated genomes was used to practice and fine-tune the technologies employed by the HGP. The genome
of the first free-living organisma strain of the Haemophilus influenza
bacteriumwas published by J. Craig Venter14 and 39 others in the July
1995 issue of Science (Fleischmann et al. 1995). Their work was the first
to use shotgun cloning, in which a whole genome is fragmented, cloned,
sequenced, and reassembled computationally through the identification of
overlapping sequences (Fleischmann et al. 1995). In doing so, individual reads are assembled into one contiguous (or contig) sequence (see
Figure 2.11). Just a year later, the whole genome sequence of the first
eukaryotic organismSaccharomyces cerevisiaewas also published in
Science (Goffeau et al. 1996). The sequencing of other significant model
organisms15 followed soon after.
Whole genome sequencing really took off in 2005, with the advent
of Next Generation Sequencing (i.e., NGS, or more generally, Next
Gen) methods. 454 Life Sciences published a sequencing-by-synthesis method16 that year in Nature (Margulies et al. 2005). This approach
involves the direct measure of released pyrophosphate molecules as single nucleotides are added to a growing DNA strand.17 Doing so mitigates
the need for terminator nucleotides and labor-intensive sequencing gels.
Sequencing-by-synthesis methods are described in more detail in Chapter 2. Around the same time, George Churchs lab from Harvard Medical
School described a multiplex polony sequencing method18 in their 2005
Science publication (Shendure et al. 2005). Both the sequencing-by-synthesis and multiplex polony methods resulted in significant savings, due
to reduced reaction volumes and enhanced throughput. Authors of the
multiplex polony strategy cited a one-ninth reduction in cost per base,
as compared to conventional methods (Shendure et al. 2005). As Next
Gen sequencing methods have evolved to increase accuracy and sequence
read lengths, the cost of genome sequencing has plummeted. If the HGP
Introduction9
had been done in 2015, the cost would have been just $1,500, assuming
that the same rate of technological innovation occurred in the absence of
the HGP-fostered collaboration of the 1990s and early 2000s (Institute
2016b). Biotechnologists today work to actualize a $1,000 human genome
sequencethe price required to routinely use whole genome sequencing
in the clinic.
1.2.2.3Organismal CloningAdvent of Stem Cell and
Transgenic Animal Biotechnologies
As the field of modern genetics blossomed in the 1950s, the prospect of
cloning whole organisms became both appealing and possible. Organismal cloning can be achieved by three primary means: (1) the splitting of
a single embryo into two identical embryos,19 (2) the fusion of an embryonic cell with an unfertilized and enucleated egg, or (3) the transfer of
all nuclear contents from an adult or embryonic somatic cell to an unfertilized, enucleated egg (see Figure 1.1). The latter, also called somatic20
cell nuclear transfer (SCNT), is the most powerful technique. In either
embryonic fusion or SCNT, the recipient egg is reactivated, and grown in
vitro to form a multicellular embryo. A cloned embryo is implanted into a
surrogate mother, or used for medicinal purposes.
(a)
(b)
Donor
embryo
Donor embryo
Recipient
egg
Split
embryos
(d)
(c)
(Embryo
fusion)
(Nucleus
removed)
Nuclear
donor
Reproductive
cloning
(Nucleus
transferred)
Cloned cell
Recipient
egg
(Nucleus
removed)
Cloned
fusion cell
Cloned cell
Cloned
embryo
Implant into
surrogate mother
Birth of cloned
animal
Cultivation of
embryonic stem
cells
Directed
differentiation
into engineered
tissues
Therapeutic
cloning
10 MODERN BIOTECHNOLOGY
Introduction11
This lambnamed Pollywas created from the nucleus of a tissue culture cell in which the human Factor IX gene was inserted. As a result,
Polly produced human Factor IX, a blood clotting protein, in her milk.34
A 2002 report in Science announced the production of pig clones that
had been modified to express human-like cell surface markers (Lai et al.
2002). Xenotransplantation (i.e., tissue transplantation between species)
is a potential medical application of such humanized animals. In 2003,
GM and cloned dairy cows were created. These cows produce milk with
enhanced protein content (Brophy et al. 2003). Cloned goats, genetically
modified to produce spider silk proteins in their milk, captured headlines
and imaginations in the early 2000s. The goal of silk milk goats was
the large-scale harvest of spider silk for the production of extraordinarily
strong, elastic, and lightweight textiles35 (Majumder, Kaulaskar, and Neogi
2015). This BioSTEEL textile could be used for bulletproof clothing or
surgical sutures.
With over a dozen animal species cloned since the late 1990s (Verma
etal. 2015), what about primates? As early as 1997, rhesus monkey
embryos were successfully cloned by nuclear transfer from blastomeres36
into enucleated eggs (Meng et al. 1997). Two live clones were born
Ditto and Netias a result of three pregnancies and 29 implanted embryos
(Meng et al. 1997). While the birth of live nonhuman primate clones made
by adult cell SCNT has not yet been actualized, multiple pregnancies have
been reported (Sparman, Tachibana, and Mitalipov 2010).
It is generally accepted that human reproductive cloning has not been
done, although there is no international or U.S. federal law prohibiting it.
12 MODERN BIOTECHNOLOGY
Introduction13
This work was a pivotal proof-of-principle for the development of patientmatched embryonic stem cells. In 2008 French et al. went further by creating human blastocysts by SCNT using adult donor nuclei (French etal.
2008). And finally in 2013, the first confirmed patient-matched human
embryonic stem cells were made using adult fibroblast cells as nuclear
donors (Tachibana et al. 2013). Collectively, this work holds immense
promise for the development of personalized embryonic stem cell therapies for human patients.
While the primate therapeutic cloning technology advanced, so did the
equally promising field of stem cell reprogramming. In 2006, Yamanaka
and colleagues were the first to transformor reprogramadult mouse
cells into those relatively indistinguishable from embryonic stem cells
(Takahashi and Yamanaka 2006). They did so through the expression of
what came to be known as the Yamanaka factorstranscription factors
Oct4, Sox2, Klf4, and Myc. These transcription factors regulate genes that
cause cells to undergo a sort of amnesia in which they forget their cellular identity (Takahashi and Yamanaka 2006). The new cells that Yamanka
et al. created by introducing and expressing the Yamanka factors were
called induced pluripotent stem cells (iPSs) (Takahashi and Yamanaka
2006). As little as two years later, human iPSs were created by a similar procedure (Park et al. 2008). Since then, iPSs have been successfully
reared into a variety of cell types and even used to populate bioscaffolds
for the growth of whole organs. The iPS technology bypasses controversial human embryo creation and destruction in the production of malleable
patient-matched stem cells for regenerative medicine.
1.2.2.4 CRISPRRevolutionizing Genome Editing
A new form of genome editing (i.e., the site-directed deletion, addition,
or insertion of chromosomal DNA by nuclease enzymes)CRISPR/
Cas9is the subject of the current biotechnological leap. CRISPR
stands for clustered regulatory interspaced short palindromic repeats,
referring to the tiny stretches of viral DNA that some bacteria carry
in their genomes (Zhang, Wen, and Guo 2014). These viral snippets,
and the proteins associated with them, make up a primitive adaptive
immune system, allowing bacteria to recognize and destroy invading
viral DNA. Naturally occurring CRISPR systems were first reported
in 1987, when researchers stumbled upon an oddly repetitive series
of sequences in a bacterial gene (Pennisi 2013). It wasnt until 2005,
when sequence comparisons revealed similarities between these kinds
of repetitive elements and viral genomes (Pennisi 2013). Not long
14 MODERN BIOTECHNOLOGY
Introduction15
16 MODERN BIOTECHNOLOGY
Index
A
Acellular subunit vaccines, 78,
8082
Acquired immunity, 7677
Activators, 25
Active vaccination, 7780
Adjuvants, 80
AduAdvantage salmon, 9394
Advanced Cell Technology (ACT),
12
Affinity tags, 7
Agrobacterium tumefaciens, 95
Algal-derived biodiesel, 110
Alleles, 21
Amino acids, 29
Aneuploidy, 58
Annealing process, 20
Annotation, 49
Anti-codons, 22
Antibody-based bioassays
ELISA, 4647
western blots, 4546
Antibody-mediated neutralization,
69
Antigens, 76
Artificially derived stem cells,
2930
Attenuated vaccines, 78
B
B cells, 76
Bt corn, 89
Bacteria, 2
Basic Local Alignment Search
Tool (BLAST), 5253
Behavior-modification strategy, 57
Bioassays, 2
Bioaugmentation, 105106
Biodiesel, 109111
Bioethanol, 108109
Biofuels
biodiesel, 109111
bioethanol, 108109
needs for, 107108
Bioinformatics, 5253
Biological molecule identification
DNA sequences, 4344
proteins, 4547
transcripts, 4445
Biological parts, 2
DNA, 1921
eukaryotic cells, 18
gene regulation, 2425
membranes, 17
prokaryotic cells, 1718
protein production, 2224
proteins, 2629
RNA, 2122
RNA templates, 26
stem cells, 2930
stock/flow system, 18, 19
three-component nucleotide
structure, 19, 20
transcription, 19, 2122
150Index
translation, 19, 22
Biomarkers, 60
Bioremediation, 104106
Bioreporters, 102104
Biosensors, 8992
3D matrices, 92
disk readers, 92
label-based detection, 90
label-free detection, 9091
miniaturization, 92
performance, 9192
receptor, 90
transducer, 90
Biostimulation, 105
Biotechnology
agricultural engineering, 4
bacteria and yeast, 2
biochemistry, 2
biological parts (see Biological
parts)
categories, 2
CRISPR system, 1314
definition, 1
disease (see Disease)
disease causation, 6062
DNA sequencing, 79
food production (see Food
production)
genetic engineering, 1
industrial fermentation, 4
interdisciplinary collaboration,
116118
organismal cloning, 913
penicillin discovery, 4
pollution (see Pollution)
public distrust, 115116
recombinant DNA technologies,
57, 82, 85, 96, 113, 120
scientific and ethical limitation,
34
viruses, 2
wicked problems, 1416
Bluewhite screening, 38
Blunt cutters, 36
Bomb-sniffing plants, 103
C
Cancer vaccines, 8384
Candidate diagnostics, 63
cDNA, 26
Cell-free fetal DNA (cfDNA), 65
Cell-mediated response, 77
Cellulosic bioethanol production,
108109
Chemical synthesis, 3334
Codons, 22
Complex multifactorial disorders,
5760
Computer modeling, 5354
Computer-aided design (CAD)
program, 5354
Contig, 42
Coverage, 48
CRISPR system, 1314
genome editing, 3940
synthetic guide RNA, 39
transgenesis, 98
Cytokines, 76
D
de novo synthesis, 33
Defense proteins, 27
Denaturation, 20
Diagnostics/Screens, 2
Differentiation, 29
Disease, 114
genetic disorders, 5760
infectious diseases, 5657
molecular diagnostics, 6266
molecular mechanisms, 6062
prevention, 7584
progression, 84
therapeutics, 6675
Disease-resistant animals, 9293
Distillers grain, 108
DNA
chemical synthesis, 3334
fingerprinting, 66
microarray, 50
polymerase chain reaction, 3033
sequence identification, 4344
Index 151
DNA ligase, 36
DNA sequencing
automated sequencers, 7
Human Genome Project, 78, 116
Sangers technique, 7, 4042
sequencing-by-synthesis method,
89, 4243
shotgun cloning, 8
whole genome sequencing, 8
Drug discovery, 7375
Druggability, 67
Duchennes muscular dystrophy
(DMD), 62
E
Ebola virus infection, 70
Edible vaccines, 8182
Electroporation, 3637
Endomembrane, 18
Endotoxin, 17
Environmental degradation.
SeePollution
Environmental monitoring,
102104
Enzyme-linked immunosorbant
assay (ELISA), 4647
Enzymes, 27
Epithelial growth factor receptor
(EGFR), 5859
Eukaryotic cells, 18
ExPASy Bioinformatics Resources
Portal, 53
F
Feedstock, 108
Food production, 114
biosensors, 8992
disease-resistant animals, 9293
modern industrialized
agriculture, 8788
transgenic crop-production,
8889
transgenics, 9397
G
Gain-of-function mutations, 58
Gel electrophoresis, 3233
GenBank, 52
Gene cloning, 57
clone transformation and
selection, 3639
CRISPR/Cas9, 3940
recombinant plasmid
construction, 3436
Gene ontology, 49
Gene regulation, 2425
Gene stacking, 98
Gene therapy, 7072
Genes, 21
Genetic disorders, 5760
Genetically modified organisms
(GMOs), 2
Genome, 21
Genomics, 4849
Global warming, 101
Guide RNA (gRNA), 39
H
Heat shock, 3637
Herceptin, 70
His-tag, 7
HIV, 7172
Homologs, 26
Housekeeping genes, 25
Human Genome Project (HGP),
78, 116
Human growth hormone, 68
Human reproductive cloning, 12
Humoral response, 77
Humulin, 6
I
Immuno-PCR diagnostics, 65
Immunoglobulins, 6970
Indirect ELISA, 4647
Induced pluripotent stem cells
(iPSs), 13, 30
152Index
Index 153
reproductive cloning, 10
somatic cell nuclear transfer,
910
therapeutic cloning, 10
xenotransplantation, 11
Orphan diseases, 5960
P
p53, 58
Palindrome, 35
Passive vaccination, 77, 78
Penicillin discovery, 4
Peptidoglycan, 17
Phenotype, 21
Photobioreactors (PBRs), 110
Phytoremediation, 106
Plasmids, 5, 34
accessory genes, 34
blunt cutters, 36
copy number, 35
isolated plasmids, 35
multiple cloning sites, 35, 36
promoters, 35
restriction enzymes, 3536
sticky ends, 3536
Pollution, 114
bioremediation, 104106
bioreporters, 102104
EPA Superfund sites, 102
global industrialization, 101
global warming, 101
liquid fuels, 107111
pollutants, 101
Polymerase chain reaction (PCR), 5
DNA Pol, 31
DNA replication, 31
DNA sequence identification, 43
gel electrophoresis, 3233
limitation, 33
temperature adjustments, 3132
transcript identification, 44
uses, 3031
Polypeptide, 22
Pre-implantation genetic diagnosis
(PGD), 66
Prokaryotic cells, 1718
Promoters, 21, 24
Prostatic acid phosphatase (PAP),
84
Protein Basic Local Alignment
Search Tool (BLASTp), 53
Proteins
ELISAs, 4647
functional categories, 27
homologs, 26
primary structure, 28
production, 2224
quaternary structure, 29
secondary structure, 2829
tertiary structure, 29
trafficking, 22
western blots, 4546
Proteomics, 51
Proto-oncogenes, 58
Provenge, 84
Purified recombinant proteins,
6869
Pyrosequencing, 4243
Q
Quantitative RT-PCR (qRT-PCR),
44
R
Recombinant DNA technologies
Asilomar Conference, 6
biopharmaceuticals, 67
guidelines, 6
plasmids, 5
polymerase chain reaction, 6
Pseudomonas strain
modification, 6
purification, 7
restriction enzymes, 5
154Index
Recombinant Ti plasmid, 96
Regenerative medicine, 7273
Regulatory proteins, 27
Renewable fuels, 108
Replication bubble, 31
Replication fork, 31
Reporter fusions, 4445
Repressors, 25
Reproductive cloning, 10
Restriction enzyme screens, 3839
Reverse transcriptase (RT), 26
Ribosomal RNA (rRNA), 22
Ribosome binding sites (RBS), 22
RNA, 2122
RNA polymerase (RNA Pol), 21
RNA silencing, 25
Roundup Ready soybean, 88
S
Sandwich ELISA, 47
Sangers technique, 7, 4042
Scaffold, 49
Sensory proteins, 27
Sequencing-by-synthesis method,
89, 4243
Shotgun cloning, 8
Signal amplification-based
diagnostics, 65
Signal proteins, 27
Single nucleotide polymorphism
(SNP), 66
Small interfering RNA (RNAi), 61
Small molecular weight
compounds (SMOLs), 66
Somatic cell nuclear transfer
(SCNT), 910, 72
Southern blot, 4344
Stem cells, 2930, 7273
Stock/flow system, 18, 19
Stop codon, 22
Storage proteins, 27
Structural proteins, 27
Subunit vaccines, 78
Surface plasmon resonance (SPR),
91
Sustainable fuels, 108
T
T cells, 7677
Target amplification, 64
Terminator nucleotides, 7
Therapeutic cloning, 10
Therapeutics
biological targets, 6667
drug discovery, 7375
gene therapy, 7072
monoclonal antibodies, 6970
purified recombinant proteins,
6869
regenerative medicine, 7273
SMOLs, 66
Thermocycling, 31
Threading, 2829
Three-component bioreporter
system, 104
Three-component nucleotide
structure, 19, 20
TinkerCell, 54
Totipotent, 29
Transcription, 19, 2122
Transcriptomics, 4951
Transcripts identification, 4445
Transfer RNA (tRNA), 22
Transgenic crop-producing, 88
Transgenics
AduAdvantage salmon, 9394
animals, 9799
animals and plants, 9495
crops, 9597
transgene protection, 97
Translation, 19, 22
Tumor suppressor genes, 58
Index 155
U
Unbiased diagnostics, 63
United Nations adopted a
Declaration on Human
Cloning, 12
Unzipping, 20
V
Vaccination and immune responses
acellular subunit vaccines, 78,
8082
acquired immunity, 7677
active vaccines, 7780
antigens, 76
development, 8284
passive, 77, 78
Vaccines, 2
Virulence factors, 56
Viruses, 2
W
Western blots, 4546
Whole cell vaccines, 7879
Whole genome sequencing, 8, 48
X
Xenotransplantation, 11
Y
Yamanaka factors, 13, 30
Yeast, 2
Z
ZMapp, 70