MODERN

BIOTECHNOLOGY

MODERN
BIOTECHNOLOGY
Defining and Solving
HumanProblems

STEPHANIE STOCKWELL, PhD

MOMENTUM PRESS, LLC, NEW YORK

Modern Biotechnology: Defining and Solving Human Problems

Copyright Momentum Press, LLC, 2017.
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Printed in the United States of America

For my beloved husband and childrenMike, Birch, and Fennawho

each made sacrifices for the sake of this book. In loving memory of
Baxter, my patient and faithful writing companion.

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

5.4 Better Liquid Fuels

5.5 Brief Summary

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.

Figure 2.1.Central dogma of biology as a stock/flow

system diagram.

19

Figure 2.2. Three-component nucleotide structure.

20

Figure 2.3.Structure of poly- and mono-cistronic genetic units and

their associated transcripts and proteins.
23
Figure 2.4. Proteins are folded polymers of amino acids.

28

Figure 2.5. Sample polymerase chain reaction (PCR) conditions.

32

Figure 2.6. Standard agarose gel for DNA electrophoresis.

33

Figure 2.7.Clones are screened by restriction enzyme fragment

analysis.

38

Figure 2.8. CRISPR/Cas9-mediated genome editing.

40

Figure 2.9. Structure of two antigen-specific antibodies.

45

Figure 2.10. Standard antibody-based bioassays.

47

Figure 2.11. Reads assembled into a consensus sequence.

49

Figure 3.1. Knockout construction by allelic replacement.

61

Figure 3.2. Simple model of a hypothetical biological system.

61

Figure 3.3. Mechanisms of antibody-mediated neutralization.

69

Figure 3.4. Common vaccine platforms.

77

Figure 3.5. System diagram of disease progression.

84

Figure 4.1. Biosensor with an optical transducer.

91

Figure 4.2. Recombinant Ti plasmid for transgenesis in plants.

96

Figure 5.1. Simple three-component bioreporter system.

104

List of Tables
Table 1.1. General categories of biotechnologies

Table 2.1. Broad functional categories of proteins

27

Table 3.1.Criteria to be optimized in the development of

molecular diagnostics

64

Table 4.1. Examples of transgenic animals and plants

94

Table 6.1.Biotechnologies to address problems of disease,

food production, and environmental degradation

114

List of Boxes
Box 1.1. Misconceptions about organismal cloning

11

Box 1.2. Challenge integration exercise: Timeline

14

Box 2.1. Gel electrophoresis

32

Box 2.2.Determining insert orientation using a restriction

enzyme screen

38

Box 3.1. Important diseases

59

Box 3.2. Molecular diagnostics in reproductive medicine

65

Box 3.3. A novel biopharmaceutical to treat Ebola virus infection

70

Box 3.4. A cure for HIV?

71

Box 3.5. The price of precision

74

Box 3.6. Challenge application exercise: Product portfolios

75

Box 3.7. Vaccines do NOT cause autism

79

Box 3.8.Challenge application exercise: Vaccine information

sheets81
Box 3.9. Edible vaccines

81

Box 3.10. Cancer vaccines

83

Box 3.11.Challenge integration exercise: System diagram of

disease progression

84

Box 4.1. Problems with industrialized food production

88

Box 4.2. Using viruses to preserve food

93

Box 4.3. Transgene protection

97

Box 4.4. Meat without livestock

99

Box 5.1. Challenge application exercise: EPA Superfund sites

102

Box 5.2. Bomb-sniffing plants

103

xvi List of Boxes

Box 5.3. Renewable versus sustainable

107

Box 5.4. Trash to treasure

109

Box 5.5. Benefits of microalgal biofuels

110

Box 6.1. Current and future biotechnology careers

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

Table 1.1. General categories of biotechnologies

Kind of biotechnology
Bioassays
Diagnostics/Screens
Vaccines
Biological Parts

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.

1.2MILESTONES IN THE DEVELOPMENT OF

MODERN BIOTECHNOLOGY
1.2.1 EARLY BIOTECHNOLOGY
Biotechnology dates back to the Ancient Egyptians. With no knowledge
of the biology governing it, these peoples performed a primitive biotechnologythe controlled fermentation of sugars for the production of
alcohol and the leavening of bread. More informed, yet still early, biotechnology emerged at the end of the 18th century (i.e., 1796) when Edward
Jenner realized the first documented immunization. He demonstrated the
power of previous cowpox exposure for the protection against small pox
infection. Louis Pasteur followed up on this work nearly 100 years later
by debunking spontaneous generation in support of the germ theory of
disease. He also developed the first artificially attenuated vaccines5 in
the late 1800s. However it wasnt until 1919 that the term biotechnology
was introduced in the written work of Kroly Ereky, a Hungarian agricultural engineer and Minister of Food. In his Biotechnology of Meat, Fat
and Milk Production in Agricultural Large-Scale Farm, Ereky described
biotechnology as the biological conversion of raw materials into u seful
products (Hollo 2000). For example, Ereckys contemporary described
the industrial fermentation of corn starch by Clostridium acetobuylicum
to yield acetone (Moses, Cape, and Springham 1999). Not long after,
Alexander Fleming accidentally discovered a fungal molecule that killed
bacteria. This molecule was further purified into penicillin, making it the
first antibiotic used to treat bacterial infections in humans. This miracle
drug arrived on the scene of war-time clinics in 1940 and saved countless
lives (Thieman and Palladino 2012).

Introduction5

1.2.2THE HISTORICAL DEVELOPMENT OF

REVOLUTIONARY BIOTECHNOLOGIES
Like all scientific fields, biotechnology is built on small incremental
discoveries, made by many researchers. Looking back, we can identify significant turning pointsmost often marked by the development
of new technologies that open doors to new applications. Most notably,
these have included the technologies associated with recombinant DNA
(rDNA), DNA sequencing, and organismal cloning. In all cases, scientists
first asked, Can we? and then Should we?
1.2.2.1Recombinant DNA TechnologiesAdvent of Gene
Cloning and Genetically Modified Organisms
Although the concept of genetic engineering was discussed among scientists as early as 1936, it was the famous solving of the double helix structure
of DNA by Watson and Crick6 in 1953 that paved the way for gene-spliced
rDNA in the early 1970s. At this time, Herbert Boyer was working to characterize a primitive bacterial immune response in which cellular enzymes
cut newly introduced (e.g., viral) DNA at predictable sites (Institute 2000
2004a). These DNA-cutting proteins were called restriction enzymes. In
1972, Paul Berg applied these enzymes to pioneer a cut-and-paste method
to genetically splice DNA (Institute 20002004b).7 These first rDNA
molecules were transferred into the fast-growing bacterium, Escherichia
coli, through the natural infectivity of lambda bacteriophage8 (Institute
20002004b). At this time, Cohen was exploring naturally occurring nonchromosomal fragments of bacterial DNAcalled plasmidsthat occasionally transfer between cells (Institute 20002004b). Cohen and Boyer
came together in 1973 to discover that the restriction enzyme, EcoRI, cuts
the E. coli plasmid pSC101 at a single site (Institute 20002004b). Here
they inserted a gene for tetracycline resistance, and used it to make a previously sensitive E. coli strain resistant to the antibiotic (Institute 2000
2004b). In doing so, they demonstrated that DNA from one source could
be cut, pasted, introduced, and propagated to transform the characteristics
of a recipient cell. Cohen and Boyer later showed that genes could even be
transferred and propagated across domains9 (Institute 20002004b).
By the mid-1970s, it became increasingly clear that genetic engineering was possible. With concerns about the implications of a technology
that he had helped create, Paul Berg convened a small group of scientists.
These scientists wrote an open letter calling for a temporary halt on rDNA

6 MODERN BIOTECHNOLOGY

research until an international committee had assessed risks and developed

responsible operating procedures/policies. This letter, thereafter called the
Berg Letter or Moratorium Letter, was published in Science on July
24, 1974, and marked the first voluntary moratorium of the scientific community. As a result, the Asilomar Conference on rDNA molecules was
convened in 1975. The group consisted of scientists, lawyers, and journalists from around the world. Recommendations were delivered to the U.S.
National Institutes of Health (NIH), where official guidelines were written
and issued the following year. Guidelines discussed proper physical and
biological containment of rDNA and the organisms that harbor them.10
Scientists continue to self-regulate rDNA research using the evolving
guidelines of the NIH.
Following the moratorium, rapid advances in rDNA technologies
transformed the landscape of molecular biology, microbial genetics, and
biotechnology. In 1983, the groundbreaking technique, polymerase chain
reaction (PCR) was developed by Kerry Mullis to replicate (or amplify)
target DNA in the absence of a cell. PCR revolutionized and empowered
both DNA sequencing and cloning, and remains one of the mostif not
the mostinfluential biotechnologies. PCR will be discussed in detail in
the next chapter.
By the late 1970s, the question of Who owns life? became a pressing legal matter. That is, can genetic material that is derived from nature
but isolated, characterized, and perhaps manipulated by scientistsbe
someones property? As early as 1873, Louis Pasteur obtained a U.S. patent for the use of pure (and naturally occurring) yeast cultures for brewing
beer (Pasteur 1873); but what about genetic material and organisms modified by rDNA technologies? This question was answered in the case of
Diamond v. Chakrabarty in 1980. Chakrabarty was a scientist working at
General Electric who sought to patent a modified Pseudomonas bacterium
that harbored plasmids conferring the ability to breakdown crude oil (Bud
1993) (1980). The U.S. Supreme Court ruled in favor of Chakrabarty, stating that the new Pseudomonas strain was indeed patentable, as it did not
occur naturally in the environment (Bud 1993). In 1988, the first transgenic11 mouse was patented in the United States by Harvard University
(Bud 1993). This set the precedent that mammalian organisms can be
intellectual property. While the United States remains the most liberal in
the patenting of life, other countries have, for the most part, followed suit.
The advent of rDNA technologies paved the way for biopharmaceuticals12medical therapeutics that are manufactured by and purified from
GM organisms. The first of these was biosynthetic human insulin, which
hit the market in the fall of 1982 (Chance and Frank 1993). Humulin

Introduction7

is biomanufactured by and purified from E. coli cells that contain the

human alpha- and beta-chain insulin encoding genes. Humulin has been
a highly successful insulin replacement therapy for hundreds of thousands
of diabetic patients.
Purification of recombinant proteins is facilitated by affinity tags
signature sequences engineered into recombinant proteins to make them
bind, and thus be captured by, other molecules. The first of these was
described in a 1988 Nature publication, and consists of six consecutive
histidine residues (commonly referred to as a His-tag) that confer specific binding affinity for nickel ions (Hochuli 1988). This affinity allows
for the separation of His-tagged proteins from complex cellular mixtures
with relative ease. Biomanufactured and purified proteins now comprise
the essential components of many medical therapeutics, vaccines, and
molecular diagnostics. Specific examples are explored in Chapter 3.

1.2.2.2 DNA SequencingAdvent of the Genomics Era

DNA sequencing is a powerful tool used to determine the exact chemical
make-up (i.e., nucleotides A, T, G, C) of purified genetic material.
The first DNA sequencing methods were independently developed by
Gilbert-Maxam and Sanger in 1980. In the Gilbert method, DNA is radiolabeled while being replicated in E. coli, carefully fragmented, and separated for size analysis. The Sanger, or terminator, method has been more
widely adopted, and therefore is described in detail in Chapter 2 (see 2.2.7).
Sangers technique involves the in vitro replication of DNA in the presence of radiolabeled dideoxynucleotides, also called terminator nucleotides. They get this name because they terminate strand synthesis when
incorporated into DNA. In 1986, Leroy Hood updated the Sanger method
by substituting fluorescent, in lieu of radioactive, labels of the terminator
nucleotides (Institute 20002004b). Doing so collapsed sequencing into
a single reaction, the products of which are detected by computer-aided
lasers and fluorescent sensors (Institute 20002004b).
The development of automated sequencers ushered in a new biotechnological eragenomicsby making the sequencing of the human genome
possible. The highly publicized and federally-funded Human Genome
Project (HGP) began in October 1990 and was estimated to take 15 years
and cost three billion dollars (Institute 20002004b). Just 11 years later,
the U.S. White House celebrated the completion of the whole human
genome when two draft sequences were published in Nature and Science
(Venter et al. 2001; Lander et al. 2001). By 2003 the draft was polished

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

Figure 1.1. Forms of organismal cloning.

10 MODERN BIOTECHNOLOGY

When the final product of the cloning procedure is the embryo, or

the stem cells that can be grown from it, the process is called therapeutic cloning (see Figure 1.1). Stem cells are undifferentiated cells that
play critical roles in growth, development, and tissue repair throughout
the body. Stem cells may differentiate into new, more specialized, cell
types. When this occurs they self-renew by asymmetrical cell division.
One daughter cell remains a stem cell and the other differentiates into
a specific cell type (e.g., nerve, skin, lung). The elasticity of a stem cell
is described in terms of potency. Stem cells that are the progenitors of
many cell types are pluripotent.21 Pluripotent stem cells like those found
in embryos are the holy grail of regenerative medicine because they can
be used to study rare diseases, heal wounds, replace dying cells, or even
grow replacement organs. The biology of stem cells is discussed in detail
in Chapter 2.
When an embryo derived by SCNT is implanted into a surrogate
mother for further development into a whole organism, it is called reproductive cloning (Niemann and Lucas-Hahn 2012). The cloned animal
that results is genetically identical to the donor nucleus from which it is
derived. Despite this, there are differences between the cloned and original animal. Most notably, the two are age-matched, due to asynchronous
gestation periods.
The first successful SCNT was accomplished in 1952 by Robert Briggs
and Thomas King (Gurdon 1997). They produced a cloned tadpole by replacing the nucleus of a leopard frog egg with that of an early-stage embryo
(Gurdon 1997). In their work to follow, they noted two things: many cloned
embryos developed abnormally, and the more differentiated (i.e., mature)
the donor nucleus, the less successful the cloning (Gurdon 1997).
The technology progressed incrementally from there, with organisms
such as rabbit (in 1975), mouse (in 1981 and 1983), sheep (in 1986), pigs
(in 1989), and cow (in 1994) (reviewed in (Gurdon and Byrne 2003)).
Along the way, based on the findings of Briggs and King, researchers
assumed that viable offspring from reproductive cloning could only be
achieved using donor nuclei from early embryonic cells. This assumption
was debunked in 1997, with the birth of Dolly,22 the cloned sheep (Wilmut
et al. 1997). What made Dolly so special was that her donor nucleus came
from the mammary gland of an adult ewe (Wilmut et al. 1997). That is,
adult organisms couldfor the first timebe cloned. This accomplishment was highly publicized and sparked a widespread debate pertaining to
the ethics and implications of organismal cloning.
Not long after the birth of Dolly, organismal cloning took another
leap when the first GM animal clone was born (Schnieke et al. 1997).

Introduction11

Box 1.1. Misconceptions about organismal cloning

Organismal cloning is a new technology.23

Clones are always artificial.24
All clones are abnormal and die young.25
Animal cloning is rare.26
Milk and meat from cloned animals is excluded from the U.S.
food supply by law.27
The first-ever animal clone was Dolly the sheep.28
A clone is the same age as the original organism.29
Clones are exactly identical in every way.30
The same procedure is used to clone all animal species.31
Human cloning is prohibited by international law.32
Extinct animals, such as mammoths or dinosaurs, have been
cloned.33

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

In 2005 the United Nations adopted a Declaration on Human Cloning, in

which members agreed to prohibit any human cloning that was incompatible with human dignity and the protection of human life (Nations 2005).
Member nations have been unable to come to a consensus on what exactly
these conditions mean in practical terms (Nations 2005). In the United
States, reproductive and therapeutic human cloning are regulated at the state
level. The 13 states that currently ban human reproductive cloning include
Arkansas, California, Connecticut, Iowa, Indiana, Massachusetts, Maryland, Michigan, North Dakota, New Jersey, Rhode Island, South Dakota,
and Virginia (Ayala 2015). In other states, privatebut no publicfunding
may be used for research involving human cloning or human stem cell lines
established after August 9, 2001 (Radio 2016; Ayala 2015).
The history of human cloning is complicated by veiled, falsified, and
unsubstantiated claims. In 1999, Advanced Cell Technology (ACT), a leading biotech company at the time, announced the creation of the first cloned
humancow hybrid embryos (News 1999). The details of this landmark
experiment remain unclear, but it is thought that the hybrid embryos were
made by transferring human donor nuclei into enucleated cow oocytes37
(News 1999). ACT allowed the hybrid embryos to grow for 12 days before
they were destroyed (News 1999). In 2002, CLONAIDa company
associated with a religious group that believes humans were created by
extraterrestrialsannounced the birth of the first cloned human, whom they
named Eve. Since then, the company has advertised human cloning services
to overcome infertility (Clonaid 20062009). None of CLONAIDs claims
have been substantiated by the scientific community (Institute 2016a).
In 2004, and years ahead of others in the field, Woo-Suk Hwangs
lab at Seoul National University reported the development of 11 human
embryonic stem cell lines from the SCNT of adult donor nuclei (Hwang
et al. 2004; Hwang et al. 2005). Not long after, some of Dr. Hwangs
junior colleagues accused him of falsifying data and unethically obtaining human egg donations (Sang-Hun 2014). Following an investigation,
Science retracted his articles in 2006. He was later convicted of embezzling research funds and bioethical misconduct (Sang-Hun 2014). Hwang
was dismissed from Seoul National University, but continues to perform
animal cloning. In 2005 he announced the creation of the first cloned dog,
Snuppy (Sang-Hun 2014). He now oversees Sooam Biotech Research
Foundation, a company in South Korea that offers cloning services to pet
owners.38
Real advances in primate cloning have come more gradually. In 2007,
Byrne et al. created the first primate embryonic stem cells (i.e., rhesus
macaque) derived from the SCNT of adult fibroblasts (Byrne et al. 2007).

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

after, the first functional study of a CRISPR system was published by

researchers at Danisco food company (Barrangou et al. 2007). Independent researchers from the United States and Germany, Jennifer Doudna
and Emmanuelle Charpentier, saw CRISPRs potential. Their teams
worked together to develop CRISPR into a genetic tool (Ledford 2015a;
Pennisi 2013), resulting in a landmark paper in 2012 (Jinek et al. 2012).
Since then, CRISPR/Cas9 has quickly taken over the genome-editing
scene, by outperforming the previous technologies; zinc finger and transcription activator-like effector nucleases (ZFNs; TALENs) (Ledford
2015a; Pennisi 2013).
Since CRISPR uses nucleic acids (rather than proteins) to hone in
on genomic targets, it is a financially accessible technology. Reagent
costs are just a fraction ($30 vs. $5,000) of those associated with
traditional approaches (Ledford 2015a). As such, the number of scientific articles mentioning CRISPR technology has grown exponentially
since 2013, while reports about ZFNs and TALENs have leveled off
(Ledford 2015a). CRISPR was even featured on the cover of the June
2016 issue of TIME Magazine, with the caption, The Gene Machine:
What the CRISPR experiments mean for humanity. Other recent headlines online and in print include CRISPR May Work On Way More
Diseases Than We Think (Park 2016) and Easy DNA Editing Will
Remake the World. Buckle Up (Maxmen 2015). The molecular details
of CRISPR/Cas9 and its current and future applications are explored in
Chapters 2, 3, and 4.
Box 1.2. Challenge integration exercise: Timeline
Using information from the previous sections, create a timeline of
the major historical events in the development of rDNA technologies,
DNA sequencing, organismal cloning, and CRISPR. What decades are
associated with major biotechnological advances? What else was going
on in the world as key biotechnologies emerged?

1.3 WICKED PROBLEMS

1.3.1 THE NATURE OF WICKED PROBLEMS
The modern world is faced with problems that are so complex that they
cannot be solved. These so-called wicked problems are unwieldy because
they intersect and overlap with complex systems involving huge n umbers

Introduction15

of people, opinions, and contradictory information (Kolko 2012). We all

appreciate the importance of wicked problemsfor example, disease,
hunger, poverty, and environmental degradation. Addressing these problems does not mean fixing them; it means taking steps to make them better.
Modern biotechnology has an arsenal of tools to address wicked
problems. Thanks to advances in DNA sequencing, rDNA, synthetic biology, and genome editing, the sequence of nearly any DNA sequence found
in nature can be defined, functionally analyzed, recreated, and introduced
into a new cell. Recombinant proteins and transgenic organisms can be
used to diagnose, treat, and prevent disease, improve crop yields, produce
biofuels, or clean up sites of contamination. Using organismal cloning,
whole organisms or stem cells can be created from a desirable original
or GM cell. These biotechnological products can be used to study complex
diseases, grow new organs, serve as bioreactors, or be the founders of
disease-resistant livestock herds.
1.3.2 THE NATURE OF SYSTEMS
Designing and implementing effective strategies to rectify wicked problems requires the ability to recognize and analyze complex systems.
Systems control every aspect of our livesfrom our bodies to our society to our planetyet their inherent properties make complex problems
difficult to understand. While components may vary, unifying principles
govern all systems.
1. Systems are more than a sum of their parts. Systems display
characteristics and outcomes that are difficult to predict. These are
called emergent properties.
2. Systems can be viewed at a variety of scales, and it is up to the
analyst to define the boundary of relevance. For example, one
might consider a disease at the molecular, cellular, organ, whole
organism, population, or ecosystem level.
3. Systems are composed of components (i.e., nouns, or stocks).
The components within a system interact with and influence one
another (i.e., verbs, or flows).
4. Complex systems are robust. System stability (i.e., homeostasis),
is maintained by an intricate network of balancing and reinforcing
loops.
5. Systems are dynamic. As a system changes over time, the relative abundance of each component (i.e., the stocks) within it will
change.

16 MODERN BIOTECHNOLOGY

6. Stocks are more easily measured than flows. Changes in stock

abundance can reveal changes in flow. For example, by measuring
the temperature of a room (a stock), one can deduce the rate of heatloss through the walls (a flow).
7. System changes result in delayed outcomes. For example, when
you pull the plug in the bath, the tub is not immediately empty.
Instead, it takes time to perceive the loss of water.
8. Inherent delays make systems difficult to understand. It is
tempting to conclude causation when two events occur simultaneously (i.e., they are correlated in time). A system analyst appreciates
system delays and instead looks for causes upstream ofinstead of
in synchrony withobserved events.
9. Once a system is defined, behaviors can more easily be interpreted, predicted, and perhaps influenced. Illustrated system
diagrams,39 with boxes, arrows, and other standardized symbols,
are a common tool for manual and computer-aided analysis.
These principles are the tools of systems thinking; a critical mindset in
a world of complex problems. Collectively these principles will serve as a
conceptual framework for understanding the ways in which biotechnology
is and can be used to interface with wicked problems related to disease,
food, and the environment. These are the foci of the coming chapters.

1.4 BRIEF SUMMARY

Biotechnology is the study and application of biological system components and interactions for the development of tools to address human
problems. Despite its ancient roots, intersection with many social and
natural sciences, and impact on modern society, the field of biotechnology is generally misunderstood and underappreciated. With the incremental development of tools such as rDNA technologies, DNA sequencing,
organismal cloning, and most recently CRISPR-mediated genome editing,
the field is uniquely poised to address complex problems related to health,
food, and the environment.

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

Industrialized food production,

8788
Infectious diseases, 5657
Inhibitory RNA (RNAi), 25
Intergenic regions, 21
International Genetically
Engineered Machines (iGEM),
54, 104, 117
Introns, 23
K
Knockout construction by allelic
replacement, 61
L
lac operon, 25
Landfarming, 105
Liposomes, 71
Loss-of-function mutations, 58
M
Measles, mumps, rubella (MMR)
vaccine, 79
Membranes, 17
MicroRNA, 25
Molecular diagnostics
biomarkers, 6263
candidate diagnostics, 63
characteristics, 64
immuno-PCR diagnostics, 65
reproductive medicine, 6566
signal amplification, 65
target amplification, 64
unbiased diagnostics, 63
Molecular tools
chemical synthesis, 3334
DNA sequencing (see DNA
sequencing)
gene cloning, 3440
polymerase chain reaction, 5,
3032

Monoclonal antibodies (mAbs),

6970
Motor proteins, 27
mRNA splicing, 23
Multiple cloning sites (MCSs), 35
N
Naturally occurring stem cells, 29
Next generation sequencing
(NGS), 48
Non-coding junk DNA, 21
Non-invasive genetic screens, 57
Noncommunicable diseases
chronic, 55
HIV, 55
infectious diseases, 5657
Noninvasive prenatal testing
(NIPT), 6566
Northern blots, 44
Nucleotide Basic Local Alignment
Search Tool (BLASTn), 53
O
Omic technologies
genomics, 4849
holistic and reductionist
approaches, 5152
proteomics, 51
transcriptomics, 4951
Open reading frames (ORFs), 49
Optical transducers, 91
Organismal cloning
Advanced Cell Technology, 12
forms of, 9
human reproductive cloning, 12
myths, 11
pig and lamp clones, 11
pluripotent stem cells, 10
potency, 10
primate cloning, 12, 13
rabbit and sheep, 10

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