Bth 303 genetic engineering

PROF. BALASUBRAMANIAN SATHYAMURTHY 2016 EDITION BTH – 303: GENETIC ENGINEERING
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FOR MSC BIOTECHNOLOGY STUDENTS
2014 ONWARDS
Biochemistry scanner
THE IMPRINT
BTH – 303: GENETIC ENGINEERING
As per Bangalore University (CBCS) Syllabus
2016 Edition
BY: Prof. Balasubramanian Sathyamurthy
Supported By:
Ayesha Siddiqui
Kiran K.S.
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I dedicate thI dedicate thI dedicate thI dedicate this material to my spiritual guru Shri Raghavendra swamigal,is material to my spiritual guru Shri Raghavendra swamigal,is material to my spiritual guru Shri Raghavendra swamigal,is material to my spiritual guru Shri Raghavendra swamigal,
parents, teachers, well wishers and students who always increase my moraleparents, teachers, well wishers and students who always increase my moraleparents, teachers, well wishers and students who always increase my moraleparents, teachers, well wishers and students who always increase my morale
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CHETAN ABBUR ANJALI TIWARI
AASHITA SINHA ASHWINI BELLATTI
BHARATH K CHAITHRA
GADIPARTHI VAMSEEKRISHNA KALYAN BANERJEE
KAMALA KISHORE
KIRAN KIRAN H.R
KRUTHI PRABAKAR KRUPA S
LATHA M MAMATA
MADHU PRAKASHHA G D MANJUNATH .B.P
NAYAB RASOOL S NAVYA KUCHARLAPATI
NEHA SHARIFF DIVYA DUBEY
NOOR AYESHA M PAYAL BANERJEE
POONAM PANCHAL PRAVEEN
PRAKASH K J M PRADEEP.R
PURSHOTHAM PUPPALA DEEPTHI
RAGHUNATH REDDY V RAMYA S
RAVI RESHMA
RUBY SHA SALMA H.
SHWETHA B S SHILPI CHOUBEY
SOUMOUNDA DAS SURENDRA N
THUMMALA MANOJ UDAYASHRE. B
DEEPIKA SHARMA
EDITION : 2016
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M. SC. BIOTECHNOLOGY – SECOND SEMESTER
BTH – 303: GENETIC ENGINEERING
4 units (52 hrs)
UNIT : 1 Introduction to Genetic Engineering 2hrs
Scope and importance of genetic engineering
UNIT : 2 Tools of Genetic Engineering 14hrs
Enzymes, Non-specific endo and exo nuclease, DNase, RNase. Restriction modification;
restriction endonuclease- types, nomenclature, recongnition sequence and mechanism
of action. Methylation, RNA modification. Role of kinases, phosphatases,
polynuclcleotide phosphorylase, polynucleotide kinase ligases – types and mechanism
of action
VECTORS : General characteristics of vectors , brief account of naturally occurring
plasmids promoters, MCS, Ori and maker gene-lac Z. construction of pBR 322,
pBR325, pUC18 and 19 vectors and expression vectors E.coli promoters, lac promoters,
lac promoters, trp promoters, lambda pL promoters, hybrid tac promoters, ribosome
binding site, codon selection. M13 derived vectors, Lambda based vectors, cosmids,
phagemids, minichromosomes, BAC’s, YAC’s , shuttle vectors, Ti plasmids, vectors for
animals-SV40 and Bovine papilloma virus.
UNIT : 3 Gene Cloning Strategies and Construction of Gene Libraries 14hrs
Cloning from mRNA, isolation and purification of RNA, synthesis of cDNA, Isolation of
plasmids, cloning cDNA in plasmid vectors, cloning cDNA in bactriaophage vectors.
cDNA library.
Cloning of genomic DNA: Isolation and purification of DNA, preparation of DNA,
preparation of DNA, fragments and cloning. Constriction of genomic libraries (Using
lambda gt 10 and 11 vectors ) In vitro packaging of lambda phage and amplification of
libraries
Advanced cloning strategies synthesis and cloning of cDNA, PCR amplified DNA, use of
adaptors and linkers, homopolymers tailing in cDNA cloning, expression of cloned DNA
molecule.
Selection, screening and analysis of recombinant
Genetic selection, insertional inactivation, chromogenic substances, complementation
of defined mutations, nucleic acid hybridization, screening methods for cloned libraries,

PCR screening protocols, immunological screening, restriction mapping of cloned
genes, blotting techniques, sequencing methods. Purification strategies of expressed
His-tagged proteins.
UNIT : 4 Transformation Techniques 8hrs
Purification of vectors DNA, restriction digestion, end modification, cloning of foreign
genes ( from mRNA, genomic DNA ) transformation screening, selection, expression and
preservation. Transformation and transfection techniques, preparation of competent
cells of bacteria, chemical methods-calcium phosphate precipitation methods, liposome
mediated method, physical methods-Electroporation, gene gun method. Method of DNA
transfer to yeast, mammalian and plant cells, transformation and transfection
efficiency.
UNIT : 5 Labelling and Detection Techniques 8hrs
Labeling of DNA, RNA and proteins by radioactive isotopes, non-radioactive labelling, in
vivo labeling, autoradiography and autofluorography. DNA sequencing by enzymatic
and chemical methods, Agarose gel electrophoresis, PAGE, PFGE. Methods of nucleic
acid hybridization; southern, northern and western blotting techniques.
UNIT : 6 Chemical Synthesis Of Genes And PCR 6hrs
Phosphodiester, phosphotriester and phophite ester methods, principles and strategies.
Oligonucleotide synthesis and application, synthesisof complete gene.
PCR, methodology, essential feature of PCR, primers, Taq polymerases, reverse
transcriptase-PCR, types of PCR-Nested, inverse, RAPD-PCR, RT-PCR (real time PCR),
Application of PCR.
References:
1. Nicholl D.S.T. Introduction to Genetic Engineering Cambridge (3rd Ed.) University
press.UK. 2008
2. Old R.W., Primrose S.B. Principles of gene manipulation - An introduction to genetic
engineering (5th Ed.), Blackwell Scientific Publications, UK. 1996.
3. David S L. Genetics to Gene Therapy – the molecular pathology of human disease(1st
Ed.) BIOS scientific publishers, 1994.

4. Ernst-L Winnacker, From Genes to Clones: Introduction to Gene Technology. WILEY-
VCH Verlag GmbH, Weinheim, Germany Reprinted by Panima Publishing Corporation,
New Delhi. 2003
5. Benjamin Lewis, Genes VIII (3rd Ed.) Oxford University & Cell Press,NY.2004
6. Robert Williamson.Genetic Engineering (1st Ed.) Academic Press.1981.USA
7. Rodriguez. R.L (Author), Denhardt D.T. Vectors: A Survey of Molecular Cloning Vectors
and Their Uses (1st Ed.) Butterworth-Heinemann publisher.UK. 1987
8. Ansubel F.M., Brent R., Kingston R.E., Moore D.D. et al. Short protocols in molecular
biology(4th Ed), Wiley publishers. India. 1999.
9. Sambrook J et al. Molecular cloning Volumes I, II and III. Cold Spring Harbor laboratory
Press, New York, USA. (1989, 2000)
10. Terence A Brown. Genomes, (2nd Ed.) BioScientific Publishers.UK.2002
11. Anthony JF Griffiths, William M Gelbart, Jeffrey H Miller, and Richard CLewontin
Modern Genetic Analysis (1st Ed.)W. H. Freeman Publishers.NY. 1999
12. S. B. Primrose, Richard M. Twyman.Principles of gene manipulation and genomics (7th
Ed.) John Wiley & Sons publishers.2006

UNIT: 1 Introduction to Genetic Engineering
Scope and importance of genetic engineering
SCOPE AND IMPORTANCE OF GENETIC ENGINEERING
Introduction
Over the past 12,000 years humans have gradually developed greater understanding
and control over life. Agriculture, including plant and animal husbandry, were early
important developments. Medicine also contributed to the control of life by fighting
disease and more recently through technologies to control and manipulate fertility.
Knowledge and technologies from physics and chemistry provide the tools to investigate
biological processes at a molecular and even atomic level. Late 20th century and 21st
century genetic science heralds remarkable advances in our understanding of life and
our ability to control and manipulate it for our teleological endeavours. Emerging
biotechnologies are in the foreground of modern scientific research. Evolutionary
theory, Mendel’s laws of inheritance, the discovery of DNA, the mapping of the human
genome, genetic engineering (GE) of organisms, gene therapy, synthetic biology, cloning,
stem cell therapies, epigenetics, and life extension research are theories and
technologies providing powerful new insights into the nature of life and the development
of technologies to manipulate all aspects of life. This knowledge is deconstructing and
reconstructing our knowledge of what life is and what it means to be human, and where
humans sit in the order of nature. Table 1 lists a brief selection of important milestones
in humanity’s understanding and control of life along with some loosely associated
worldviews.
Genetic technology has the potential to change biological and social reality. Its
development and application have consequences for humans, other animals and the
planetary biosphere. These consequences are open to moral evaluation, questions that
may be asked include:
What are the likely social and moral impacts? Is this progress? Are these consequences
good or bad? Does the potential good outweigh the potential bad? For whom? How fair
are theconsequences? How easily can they be accessed or avoided? And how do
different social and biophysical contexts affect their moral status? Another relevant
question is, can the positive consequences obtained by use of genetic technology be

obtained using alternative technologies (perhaps with less potential for negative
consequences)? These questions demonstrate that the practice of genetic science (and
indeed science in general) is inextricably bound to moral reasoning, moral behaviour
and technological foresighting.
The rise of genetic science
Darwin’s Theory of Evolution completely revised our notions of the nature of life and its
origins. Species were no longer created individually by God, nor once ‘created’, were
they fixed and immutable. No longer were we a unique and special creature, made in
the image of a miraculous supernatural creator, rather, it became apparent that
humans were one of approximately ten millions species inhabiting earth, evolving to fit
selection pressures in a similar fashion to the other animals on the planet. Gregor
Mendel’s laws of inheritance statistically demonstrated that characteristics could be
passed on from one generation to the next. The discovery, in the early twentieth century
by Thomas Hunt Morgan, of chromosomes and the genetic diversity engendered by
sexual reproduction, and the mid century discovery of DNA by Crick and Watson
provided a causal mechanism for inheritance and a molecular level mechanism for
Darwinian natural selection.
Biological Milestones Year Associated worldview
Agriculture – plant and animal
Husbandry
10,000BC Animistic/magical/mythological
Ancient medicine (e.g., Imhotep,
Hippocrates, Galen)
2500BC –
180AD
Animistic/magical/mythological
/religious/Ptolemaic
Medieval medicine (e.g.,Avicenna,
Ibn an-Nafis, Paracelsus)
1000-
1500AD
Religious/Ptolemaic
Renaissance medicine (e.g.,
Vesalius to Jenner)
1500-
1800
Religious/Copernican/scientific
Darwin’s Theory of Evolutionary 1860
Mendel’s Laws of inheritance 1865
Pasteur invents vaccines 1880
Morgan’ discovery of the 1915

chromosomes
Fleming invents antibiotics 1928 Religious/Copernican/scientific/
modernistWatson and Crick discover DNA 1953
Fertility control – oral
contraceptive, in vitro fertilisation
1960
Genetic engineering 1971
Tissue engineering 1987 Copernican/scientific/post
modernistGene therapy (1970)
1990
Epigentics 1990
Animal cloning 1996
Stem cells therapy 1998
Life extension 2000
Synthetic biology 2000
Technology has enabled the genomes of organisms to be ‘read’ and compared, showing
that humans share more than 98% of our genes in common with the chimpanzee
(Jones, 2006), giving us new insights into our biological and moral position within
nature.
The Human Genome Project (HGP) achieved three major goals. First, it sequenced the
order of all the 2.9 billion base pairs in the genome. Second, it developed maps locating
genes for major section of all our chromosomes. Third, it produced ‘linkage maps’
enabling inherited traits to be tracked over generations. Francis Collins, the director of
the HGP described the results and meaning of the HGP as:
It's a history book - a narrative of the journey of our species through time. It's a shop
manual, with an incredibly detailed blueprint for building every human cell. And it's a
transformative textbook of medicine, with insights that will give health care providers
immense new powers to treat, prevent and cure disease. (Cited by National Human
Genome Research Institute, 2009).
As the relationship between genes and individual health and behaviour becomes more
apparent, moral questions arise as to who may have access to an individual’s genome,
and what will they be able to do with this information. As significant a milestone as it
is, sequencing of the genome merely marks a beginning. It will take many decades (and

massive computer power) to understand how the approximately 20,000 genes in the
human genome interact with one another to produce over two hundred thousand
different proteins.
A great deal is not currently understood about how the genome works. Long held
theories continue to be questioned. For example, contrary to the last hundred years of
scientific belief, Mendel’s Laws have recently been challenged. Although still believed to
be fundamentally correct, it has been claimed that Mendel’s Laws are not absolute and
exceptions occur (Lolle, Victor, Young, & Pruitt, 2005). Likewise, the idea of inherited
acquired characteristics was for a long time considered biological and scientific heresy,
but the received scientific dogma has been challenged by the new science of epigenetics
(Jablonka & Raz, 2009; Kaati, Bygren, & Edvinsson, 2002; Lumey, 1992). Similarly, a
dozen years ago, with perhaps a little scientific arrogance, molecular biologists
designated long stretches of organisms’ genomes as “junk DNA” claiming that these
non-coding segments served no purpose. However, it is logically obvious that human
lack of knowledge about the function of elements of nature does not mean they lack
function.
Recently, research has shown important roles for junk DNA (Nowacki, et al., 2009),
demonstrating the hubris of the junk DNA assumption. Indeed, it now appears that
junk DNA plays a vital role in evolution (in particular enabling fast genetic adaptation to
changing environmental circumstances) and will be crucial for the refining of GE
techniques and for gene therapy (Feng, Naiman, & Cooper, 2009; Vinces, Legendre,
Caldara, Hagihara, & Verstrepen, 2009). New evidence also suggests that the rDNA
repeats known as “junk DNA” are essential for repairing the DNA damage caused by
factors such as UV light (Ide, Miyazaki, Maki, & Kobayashi, 2010). The use of
technologies with powerful potential to affect the physical and social worlds, without a
good understanding of the science involved, has the potential for unexpected and
unforeseen negative social and moral impacts.
Developments in genetic science and moral questions
Genetic engineering
The breeding of promising individuals over generations in order to create desirable
phenotypic characteristics in plants and animals has long been practiced in
horticulture and animal husbandry. This is a relatively slow process with progressive

changes made over many generations, not by nature or natural selection, but by human
intervention in the evolutionary progress of the species. Racehorses, domestic cattle,
show dogs and the staple grains are prime examples of centuries and even millennia of
breeding to slowly bend nature to the aesthetic tastes and teleological desires of
humans.
In the past forty years, with the discovery of recombinant DNA, humans have gained
the power to make changes to an organism’s genome in a single generation. Genetic
engineering (GE) involves the chemical addition or deletion of a specific gene from an
organism’s genome in order to bring about a desired change in the organism’s
phenotype. With this process organisms can have current characteristics enhanced or
removed and even entirely new characteristics, not evident in the organism’s species,
added. Thus, a gene from one species (or a synthetic analogue of the gene), may be
spliced into the genome of the same or a different species, or even an organism from a
different biological kingdom, giving the new GE organism phenotypic characteristics
from the donor species (Small, 2004a).
In this way GE can create organisms with desired attributes much more quickly than
traditional breeding (i.e., in a single generation). This amounts to a speeding up of
evolution in a direction decided by humans. This also differs from normal evolution and
animal and plant husbandry in that the new organism does not co-evolve, in little steps,
over time with the other organisms in its environment. Instead an evolutionary leap is
engineered within a single generation. Another difference between GE and selective
breeding is that organisms can be created that could not possibly have come about
naturally, as organisms generally cannot breed with others from different species or
kingdoms. Proponents see great hope for the common good of humanity in GE
technology, and often claim that the technology will be necessary to produce enough
food to feed the future population (Borlaug, 1997; Fedoroff, et al., 2010; Ortiz, 1998).
While GE offers the potential to further bend nature to our desires, critical
commentators express concern about negative extrinsic moral impacts. These include
the potential to develop dangerous organisms, the impossibility of reversibility once
such organisms are loose in the environment, and the potential for negative impacts on
humans, other animals and the environment (Antoniou, 1996; Fox, 1999; Ho, 2000;
Rifkin, 1998; Straughan, 1995b). Others criticise the technology from an intrinsic moral

perspective; creating life is the province of ‘God’ or nature – human attempts to usurp
the role of God or nature are seen as acts of hubris – against God or disrespectful to
nature (Appleby, 1999; Straughan, 1995a).
Currently GE is being used to engineer micro-organisms and bacteria (particularly for
the production of medicines such as insulin, factor 9 clotting agent, human growth
hormone, etc.), plants and animals for food production, production of medicines,
industrial production and phytoremediation. An example of a potential GE food animal
is the ‘ecofriendly’ GE pig, engineered to contain bacteria which help pigs remove
phosphate from their food, thus stopping it from passing through into the environment,
where it causes harm to life in streams and rivers (Golovan, et al., 2001). Pigs have also
been genetically engineered to contain human genes, so that their organs will be less
susceptible to immune system rejection when used for xenotransplantation (White,
Langford, Cozzi, & Young, 1995); the replacement of failing human organs with those
from animals.
Advocates of GE claim that the technology is safe. In 2008 GE crops were grown on 300
million acres worldwide. GE crops have been consumed for over 13 years without any
incident, it is claimed. Furthermore, production has increased and so have farmers’
profits, while pesticide and herbicide use have been reduced and the use of the no-till
method of agriculture (helpful for reducing soil erosion) increased (Fedoroff, et al.,
2010). However, so far the principal use of GE in food crops has been to engineer insect
resistance (bt crops) or to make the crops resistant to a specific herbicide used to
eliminate weeds from fields of growing crops – a major beneficiary being the company
selling the proprietary herbicide and seeds (one and the same company – Monsanto).
On the positive side, the herbicide for which resistance is engineered (Roundup or
glyphosate) is relatively environmentally benign and the whole process eliminates the
need for further applications of less environmentally benign herbicides.
One possibility presented by GE is the enhancement of nutritional qualities of crops, as
for example, the much heralded golden rice. Golden rice has been engineered to contain
extra beta-carotene which converts to vitamin A when consumed by humans. Many
people in developing countries, where rice is the primary staple, suffer from vitamin A
deficiency (Tang, Qin, Dolnikowski, Russell, & Grusak, 2009). Foods with genetically
enhanced health qualities or with healthy additives are referred to as functional foods

and the science of developing them and studying the relationship between food plant
genes, health and the individual human genome is called nutrigenomics. Of course, the
societal benefits of functional foods will be dependent upon the public’s acceptance of
GE food.
Genetic engineering for medical purposes is considerably more acceptable to the general
public than GE of food crops (Small, Parminter, & Fisher, 2005). Proponents hope that
numerous medicines will be able to be grown in GE plants and/or GE animals and
produced more cheaply than through current techniques. A biotech company,
SemBioSys, has submitted an Investigational New Drug application for safflower-
produced recombinant human insulin to the U.S. FDA (SemBioSys, 2008). Edible
vaccines (e.g., potatoes, tomatoes, bananas etc) are being developed for a range of
diseases (e.g., cholera, measles, malaria, hepatitis B, type 1 diabetes etc) and are
proposed as a logistically simpler resolution of the problem of getting vaccines to those
in need in developing countries (Chowdhury & Bagasra, 2007; Levi, 2000). However, it
remains unclear how vaccine dosages would be controlled and how accepting the public
will be of the conflation of food and medicine. Nonetheless, biotech and pharmaceutical
companies have high hopes for rich profit streams from genetically enhanced medical
foods and functional foods.
GE animals have been used as ‘bioreactors’ to produce medicines and industrial
products. Cows, sheep and goats have been genetically engineered to produce human
proteins in their milk for medical purposes (Wells, 2010). Silk worms have been
genetically engineered to produce a form of the human protein collagen which scientists
hope to harvest for applications such as artificial skin and wound dressings (Tomita, et
al., 2003).
The industrial sector also contains many potential applications for GE technology in
terms of new methods of producing currently available materials, new materials with
desirable qualities, and the production of chemicals and biofuels. For example, spider
silk is stronger than steel and as resilient as kevlar, but it is very expensive to produce.
Scientist have placed artificial versions of silk genes in various plants (potatoes,
tobacco) and animals (goats) and, using this technology, hope to be able to mass
produce silk protein for the development of new biodegradable ‘super-materials’
(Scheller, Guhrs, Grosse, & Conrad, 2001).

Gene engineered viruses have even been used to manufacture a ‘green battery’ which
the authors claim is capable of powering an iPod three times as long as current iPod
batteries (Lee, et al., 2009). However, some GE animals seem largely for human
entertainment, for example, the first GE pet commercially available in the U.S. was a
florescent red zebrafish called a GloFish (GloFish.com, 2010). A company called
Lifestyle Pets has marketed a genetically engineered hypoallergenic cat. Given the
history of animal breeding for traits of interest to humans, further such applications
seem highly probable. Indeed, GE pets suggest mythological sized possibilities; anyone
for a pet gryphon? Chimeras are indeed possible using genetic technologies, with a
number of research projects having already created them (however, a gryphon might be
a bit of a stretch). Of particular concern to some is the possibility of human-animal
chimeras (Robert & Baylis, 2003). Robert and Baylis imagine a fusion between a chimp
and a human. They suggest that there might be confusion over the status of such a
creature and that it might lead to social disorder. However, Savulescu (2003) argues
that there might be good reasons to create human chimeras. He suggests medical
reasons (e.g., to confer resistance to specific diseases such as AIDS), to delay aging, or
to enhance human capabilities.
Clearly, a range of ethical questions are opened by the creation of chimeras.
Undoubtedly, there will be a range of different responses to these questions. Another
question some ethicists have raised regarding GE animals concerns respect for the telos
of the animal. Telos refers to the “genetically based drives or instincts that, if frustrated,
would result in a significant compromise to the welfare of an animal” (Thompson, 2010,
p. 817). Some ethicists claim that it may be morally acceptable to alter an animal’s telos
using GE so long as it enhances wellbeing (Rollin, 1998), while others have argued that
it is not (Fiester, 2008).
AREA Technology /
Product
Potential Benefits Potential Harm
Food GE crops Less pesticides and
Herbicides.
Less fertilizer.
No till Agriculture
(soil conservation)
Extrinsic
Resistant pests (evolve)
Super weeds (outcrossing
and escape)
Irreversibility

Environmentally
resilient crops
Crops with enhanced
nutritional value
Single generation
evolutionary impacts
Conflation of food and
medicine
Lack of knowledge
Accidental or incidental
negative impacts on
humans, animals, and
environment
Intrinsic and emotional
Playing God
Disrespectful to nature
Morally/spiritually wrong
Emotional yuk factor
GE animals Increased production
Healthier meat
More resilient
animals
(less medicines,
increased
environmental
tolerance)
Extrinsic
Reduced species diversity
Single generation
evolutionary impacts
Conflation of food and
medicine
Lack of knowledge
negative impacts on
environment
Playing God
Disrespectful to animal
telos

Medicine Therapy
Medicines
derived from
GE
microorganisms,
plants, animals.
Gene therapy
Stems cells
Tissue
engineering
New medicines for
curing illness, and
injury
Organ replacement
Elimination of some
diseases
Increased life
expectancy
Outcrossing (and/or
escape)- Irreversibility -
Lack of knowledge
negative impacts on
environment - Zoonotic
disease (e.g. from
xenotransplantations)
Overpopulation
Malevolent actions (GE
virus developed as weapon)
Same as for GE food
animals
Enhancement
Somatic and
germline
therapy
(enhanced
physical,
social mental
capabilities, life
extension)
Chimeras
Enhanced human
(and non-human)
capabilities
Increased human
resilience
Disease elimination
Promotion of human
wellbeing
Much increased life
expectancy
Extrinsic
Super warriors - Eugenics
Lack of knowledge
negative impacts on
humans, Fairness/justice
Autonomy - Species
divergence - Potential
enforcement -
Overpopulation
Playing God - Disrespect to
nature - Disrespectful to
human telos - Morally/
spiritually wrong
Industry GE Pets, GE Pets with reduce Extrinsic

plants, animals
microorganism
for
manufacturing
Chemicals and
materials
Energy and fuels
Synthetic biology
Bioinformatics
Biomimetics
allergic potential
New and existing
chemicals and
materials with a
range of new or
enhanced properties
Mitigation of peak oil
New production
methods and
processes
Outcrossing or escape
Dangerous organisms
Irreversibility - Competition
between food and fuel for
land and water - Lack of
knowledge - Accidental or
incidental negative impacts
on humans, animals, and
environment - Malevolent
bioweapons
Same as for GE food crops
Ecosystem
services
Phytoremediation
Trees with
enhanced
carbon
absorption
Remediation of
pollution and toxic
sites
Climate change
mitigation
Extrinsic
Outcrossing or escape
Irreversibility
Lack of knowledge
Unforeseen or incidental
negative impacts on
environment
negative impacts on
environment
Malevolent application as
bioweapons
Playing God

UNIT : 2 TOOLS OF GENETIC ENGINEERING
Enzymes, Non-specific endo and exo nuclease, DNase, RNase. Restriction
modification; restriction endonuclease- types, nomenclature, recongnition
sequence and mechanism of action. Methylation, RNA modification. Role of
kinases, phosphatases, polynuclcleotide phosphorylase, polynucleotide kinase
ligases – types and mechanism of action
VECTORS : General characteristics of vectors , brief account of naturally
occurring plasmids promoters, MCS, Ori and maker gene-lac Z. construction of
pBR 322, pBR325, pUC18 and 19 vectors and expression vectors E.coli promoters,
lac promoters, trp promoters, lambda pL promoters, hybrid tac promoters,
ribosome binding site, codon selection. M13 derived vectors, Lambda based
vectors, cosmids, phagemids minichrosomes, BAC’s, YAC’s , shuttle vectors, Ti
plasmids, vectors for animals-SV40 and Bovine papilloma virus.
ENZYMES, NON-SPECIFIC ENDO AND EXO NUCLEASE, DNASE, RNASE.
DNases: Deoxyribonuclease I cleaves double-stranded or single stranded DNA. Cleavage
preferentially occurs adjacent to pyrimidine (C or T) residues, and the enzyme is
therefore an endonuclease. Major products are 5'-phosphorylated di, tri and
tetranucleotides.
In the presence of magnesium ions, DNase I hydrolyzes each strand of duplex DNA
independently, generating random cleavages. In the presence of manganese ions, the
enzyme cleaves both strands of DNA at approximately the same site, producing blunt
ends or fragments with 1-2 base overhangs. DNase I does not cleave RNA.
Some of the common applications of DNase I are:
• Eliminating DNA (e.g. plasmid) from preparations of RNA.
• Analyzing DNA-protein interactions via DNase footprinting.
• Nicking DNA prior to radiolabeling by nick translation.
Exonuclease III(E. coli): Removes mononucleotides from the 3' termini of duplex DNA.
The preferred substrates are DNAs with blunt or 5' protruding ends. It will also extend
nicks in duplex DNA to create single-stranded gaps. In works inefficiently on DNA with
3' protruding ends, and is inactive on single-stranded DNA.
Mung Bean Nuclease: Digests single-stranded DNA to 5'-phosphorylated mono or
oligonucleotides. High concentrations of enzyme will also degrade double-stranded

nucleic acids. Used to remove single-stranded extensions from DNA to produce blunt
ends.
Nuclease BAL 31 : Functions as an exonuclease to digest both 5' and 3' ends of double-
stranded DNA. It also acts as a single-stranded endonuclease that cleaves DNA at
nicks, gaps and single stranded regions. Does not cleave internally in duplex DNA.Used
for shortening fragments of DNA at both ends.
Nuclease S1: The substrate depends on the amount of enzyme used. Low
concentrations of S1 nuclease digests single-stranded DNAs or RNAs, while double-
stranded nucleic acids (DNA:DNA, DNA:RNA and RNA:RNA) are degraded by large
concentrations of enzyme. Moderate concentrations can be used to digest double
stranded DNA at nicks or small gaps.
Used commonly to analyze the structure of DNA:RNA hybrids (S1 nuclease mapping),
and to remove single-stranded extensions from DNA to produce blunt ends.
Ribonuclease A is an endoribonuclease that cleaves single-stranded RNA at the 3' end
of pyrimidine residues. It degrades the RNA into 3'-phosphorylated mononucleotides
and oligonucleotides.
RNases, which play important roles in nucleic acid metabolism, are found in both
prokaryotes and eukaryotes, and in practically every cell type. The human body uses
RNases to defend against invading microorganisms by secreting these enzymes in fluids
such as tears, saliva, mucus, and perspiration.
RNase H: RNase H (Ribonuclease H ) is an endoribonuclease that specifically hydrolyzes
the phosphodiester bonds of RNA which is hybridized to DNA. This enzyme does not
digest single or double-stranded DNA.
Applications:
Removal of poly(A) tails of mRNA hybridized to poly(dT)
Removal of mRNA during second strand cDNA synthesis
Ribonuclease If (RNase If) is a single strand specific RNA endonuclease which will cleave
at all RNA dinucleotide bonds leaving a 5´ hydroxyl and 2´, 3´ cyclic monophosphate (1).
RNase Ifis a recombinant protein fusion of RNase I (from E. coli) and maltose-binding
protein. It has identical activity to RNase I.
Applications:
Degradation of single-stranded RNA to mono-, di- and trinucleotide (3)

Used in ribonuclease protection assays
RNase A:
RNase A is a pancreatic ribonuclease that cleaves single-stranded RNA. Bovine
pancreatic RNase A is one of the classic model systems of protein science. The positive
charges of RNase A lie mainly in a deep cleft between two lobes. The RNA substrate lies
in this cleft and is cleaved by two catalytic histidine residues, His12 and His119, to
form a 2',3'-cyclic phosphate intermediate that is stabilized by nearby Lys41.
Eliminating or reducing RNA contamination in preparations of plasmid DNA.
Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in
RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be
analyzed.
RESTRICTION MODIFICATION; RESTRICTION ENDONUCLEASE- TYPES,
NOMENCLATURE, RECONGNITION SEQUENCE AND MECHANISM OF ACTION.
Restriction Modification System
Phage (or viruses) invade all types of cells. Bacteria are one favorite target. Defense
mechanisms have been developed by bacteria to defend themselves from these
invasions. The system they possess for this defense is the restriction-modificiation
system. This system is composed of a restriction endonuclease enzyme and a
methylase enzyme and each bacterial species and strain has their own combination of
restriction and methylating enzymes.
Restriction enzyme - an enzyme that cuts DNA at internal phosphodiester bonds;
different types exist and the most useful ones for molecular biology (Type II) are those
which cleave at a specific DNA sequence
Methylase - an enzyme that adds a methyl group to a molecule; in restriction-
modification systems of bacteria a methyl group is added to DNA at a specific site to
protect the site from restriction endonuclease cleavage
Several different types of restriction enzymes have been found but the most useful ones
for molecular biology and genetic engineering are the Type II restriction enzymes. These
enzymes cut DNA at specific nucleotide sequences. For example, the enzyme EcoRI
recognizes the sequence:
5' - G A A* T T C - 3'
3' - C T T *A A G - 5'

*The site of methylation protection from restriction enzyme cleavage is the 3'
adenine.This enzyme always cuts between the 5' G and A residues. But if we look at the
sequence we can see that both strands will be cut and leave staggered or overlapping
ends.
5' - G A A T T C - 3'
3' - C T T A A G - 5'
Not all Type II restriction enzymes generate staggered ends at the target site. Some cut
and leave blunt ends. For example, the enzyme BalI.
5' - T G G C* C A - 3'
3' - A C *C G G T - 5'
is cut at the point of symmetry to produce:
5' - T G G C C A - 3'
3' - A C C G G T - 5'
(Note: * The site of methylation protection from restriction enzyme cleavage; 5' cytosine)
The bacterial cell uses the restriction enzyme to cut the invading DNA of the virus at the
specific recognition site of the enzyme. This prevents the virus from taking over the
cellular metabolism for its own replication. But bacterial DNA will also contain sites
that could be cleaved by the restriction enzyme.
How is the bacterial cell protected? This protection is offered by the action of the
methylase. The methylase recognizes the same target site as the restriction enzyme and
adds a methyl group to a specific nucleotide in the restriction site. Methylated sites are
not substrates for the restriction enzyme. The bacterial DNA is methylated immediately
following replication so it will not be a suitable substrate for restriction endonuclease
cleavage. But it is unlikely that the invading viral DNA will have been methylated so it
will be an appropriate target for cleavage. Thus, the viral DNA is restricted in the
bacterial cell by the restriction enzyme, and the bacterial DNA is modified by the
methylase and is provided protection from its own restriction enzyme.
Isoschizomers are pairs of restriction enzymes specific to the same recognition
sequence.
Restriction endonucleases that recognize the same sequence are isoschizomers.

For example, Sph I (CGTAC/G) and Bbu I (CGTAC/G) are isoschizomers of each other.
The first enzyme to recognize and cut a given sequence is known as the prototype, all
subsequent enzymes that recognize and cut that sequence are isoschizomers.
HpaII (recognition sequence: C↓CGG) and MspI (recognition sequence: C↓CGG).
Neoschizomer: An enzyme that recognizes the same sequence but cuts it differently is
a neoschizomer. Neoschizomers are a specific type (subset) of Isoschizomers.
For example, Sma I (CCC/GGG) and Xma I (C/CCGGG) are neoschizomers of each
other.
Thus, AatII (recognition sequence: GACGT↓C) and ZraI (recognition sequence:
GAC↓GTC) are neoschizomers of one another,
METHYLATION
Among these mechanisms, DNA methylation, or the enzymatically mediated addition of
a methyl group to cytosine or adenine dinucleotides, serves as an inherited epigenetic
modification that stably modifies gene expression in dividing cells. The unique
methylomes are largely maintained in differentiated cell types, making them critical to
understanding the differentiation potential of the cell.
In the DNA methylation process, cytosine residues in the genome are enzymatically
modified to 5-methylcytosine, which participates in transcriptional repression of genes
during development and disease progression. 5-methylcytosine can be further
enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine
dioxygenases. DNA methylation affects gene transcription by physically interfering with
the binding of proteins involved in gene transcription.
Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that
can then recruit additional proteins. Some of these include histone deacetylases and
other chromatin remodeling proteins that modify histones, thereby forming compact,
inactive chromatin, or heterochromatin. While DNA methylation doesn’t change the
genetic code, it influences chromosomal stability and gene expression.
RNA MODIFICATION.
PHOSPHATASES
Alkaline Phosphatase is an important tool in molecular biological processes like cloning.
It removes 3’- phosphate groups from a variety of substrates. Although in laboratory, it
is used to catalyze the removal of terminal 5’-(P), residues from single stranded or

double stranded DNA and RNA. The resulting 5’ -OH termini can no longer take part in
ligation reactions, thus prevents self religation of vectors, reducing the background of
transformed bacterial colonies that carry empty plasmids. This enzyme works optimally
at alkaline pH (range of 8-9 in the presence of low Zn+2 concentrations) and hence
derived the name.
Alkaline Phosphatase is isolated from various sources:
Bacterial Alkaline phosphatase
Secreted in monomeric form into the Periplasmic space of E.coli, where it form dimers
and gets catalytically activated. It’s a remarkably stable enzyme and is resistant to
inactivation by heat and detergent. Thus, bacterial alkaline phosphatase is the most
difficult to destroy in the reaction mix.
Calf Intestinal Phosphatase
Calf intestinal phosphatase is a dimeric glycoprotein isolated from bovine intestine. This
has much more practical significance than bacterial alkaline phosphatase, since it can
be readily inactivated from the reaction mixture using proteinase K or by heating at
65˚C for 30 minutesor 75˚C for 15 minutes in the presence of 10mM EGTA.
Shrimp alkaline phosphatase
Extracted from cold water shrimp, can be inactivated readily by heating at 65˚C for 15
min.
POLYNUCLEOTIDE PHOSPHORYLASE
Polynucleotide Phosphorylase (PNPase) is a bifunctional enzyme with a
phosphorolytic 3' to 5' exoribonuclease activity and a 3'-terminal oligonucleotide
polymerase activity. That is, it dismantles the RNA chain starting at the 3' end and
working toward the 5' end. It also synthesizes long, highly heteropolymeric tails in vivo.
It accounts for all of the observed residual polyadenylylation in strains of Escherichia
coli missing the normal polyadenylylation enzyme.
It is involved on mRNA processing and degradation in bacteria, plants, and in humans.
In humans, the enzyme is encoded by the PNPT1 gene. In its active form, the protein
forms a ring structure consisting of three PNPase molecules. Each PNPase molecule
consists of two RNase PH domains, an S1 RNA binding domain and a K-homology
domain. The protein is present in bacteria and in the chloroplasts and mitochondria of
some eukaryotic cells.

POLYNUCLEOTIDE KINASE
A kinase from bacteriophage T4 that transfers the γ-phosphate of ATP to the 5' end of
DNA or RNA. Used to end label DNA and to phosphorylate synthetic DNA
LIGASES – TYPES AND MECHANISM OF ACTION
DNA ligases close nicks in the phosphodiester backbone of DNA. Biologically, DNA
ligases are essential for the joining of Okazaki fragments during replication, and for
completing short-patch DNA synthesis occurring in DNA repair process.
The smallest known ATP-dependent DNA ligase is the one from the bacteriophage T7 (at
41KdA). Eukaryotic DNA ligases may be much larger (human DNA ligase I is >
100KDA) but they all appear to share some common sequences and probably structural
motifs.
DNA Ligase Mechanism
The reaction occurs in three stages in all DNA ligases:
Formation of a covalent enzyme-AMP intermediate linked to a lysine side-chain in the
enzyme.
Transfer of the AMP nucleotide to the 5’ phosphate of the nicked DNA strand.
Attack on the AMP-DNA bond by the 3’-OH of the nicked DNA sealing the phosphate
backbone and resealing AMP.
The following figure illustrates the three reaction stages:
DNA ligases are Mg++-dependent enzymes that catalyze the formation of phosphodiester
bonds at single-strand breaks in double-stranded DNA. The first step in the reaction is
the formation of a covalent enzyme/adenylate intermediate.
The ATP is cleaved to AMP and pyrophosphate with the adenylyl residue linked by a
phosphoramidate bond to the &-amino group of a specific lysine residue at the active
site of the protein. The reaction is readily reversed in vitro by addition of
pyrophosphate.
The activated AMP residue of the DNA ligase/adenylate intermediate is transferred to
the 5 -phosphate terminus of a single-strand break in double-stranded DNA to generate
a covalent DNA-AMP complex with a 5'-5' phosphoanhydride bond. In the final step of
DNA ligation, unadenylylated DNA ligase is required for the generation of a
phosphodiester bond and catalyzes displacement of the AMP residue through attack by
the adjacent 3 -hydroxyl group on the adenylylated site.

DNA LIGASE I
It is present in all the eukaryotic cells. The main function of human DNA ligase I is
probably the joining of Okazaki fragments during lagging-strand DNA replication. The
enzyme is also involved in DNA excision repair. Similarly to DNA polymerase-A, DNA
ligase I is induced 10- to 15-fold in S phase in mammalian cells. Thus, it is present at
much higher concentrations in proliferating tissues.
DNA ligase I is a phosphoprotein, and most or all of the phosphate residues are
localized to the amino-terminal region. Furthermore, the amino-terminal part is highly
susceptible to proteolysis, so a 78-kD active fragment of mammalian DNA ligase I,
comprising the catalytic domain of the enzyme, is often generated as a preparation
artifact due to endogenous degradation during enzyme purification.
The human gene encoding DNA ligase I is located at chromosome 19q13.2-13.3. The
gene covers 53 kb and contains 28 exons. DNA ligase I is more effective at blunt-end
joining than mammalian DNA ligases I1 and 111 but is less efficient in this regard than
bacteriophage T4 DNA ligase.
DNA LIGASE II
This approximately 69-kD DNA ligase can be distinguished from DNA ligase I by its
ability to join an oligo(dT)*poly(rA) substrate. DNA ligase I1 is not induced on cell
proliferation, and its cellular role is not clear. It is the major DNA ligase activity in
certain nonproliferating tissues, e.g., adult liver.
The enzyme is more firmly retained in cell nuclei than DNA ligase I and requires buffers
of moderate or high salt concentration for efficient extraction.
DNA LIGASE III
DNA ligase 111 is a mammalian DNA ligase of 103 kD. The enzyme resembles DNA
ligase I in having a protease-sensitive amino-terminal region not required for DNA

ligation activity in vitro, but it resembles DNA ligase I1 in its ability to join an
oligo(dT)*poly(rA) substrate.
DNA LIGASE IV
A fourth mammalian DNA ligase has been detected recently by a search of expressed
sequence tags with short sequence motifs highly conserved in eukaryotic DNA ligases
(Wei et al. 1995). The complete cDNA encodes a 96-kD protein with DNA ligase activity
that exhibits partial sequence homology with ligases 1-111.
The human gene for DNA ligase IV is localized on chromosome 13q33-34. Northern
blots indicate that the enzyme is expressed in thymus and testis and at a very low level
in several other tissues, but its physiological role is not known. An unusual feature of
DNA ligase IV, in comparison with the other mammalian DNA ligases, is an extended
carboxy-terminal region of more than 300 amino acids that shows no homology with
other proteins in databases. This region may conceivably be involved in protein-protein
interactions that could functionally distinguish this enzyme from the other DNA ligases.
VIRUS-ENCODED DNA LIGASES
Herpesviruses and smaller DNA and RNA animal viruses do not encode a DNA ligase.
However, a distinct virus-encoded enzyme is produced by vaccinia virus and a number
of other poxviruses.
The 63-kD vaccinia protein only shows weak homology (-30%) with DNA ligase I from
human cells, S. pombe, or S. cerevisiae. Although poxviruses replicate in the host-cell
cytoplasm, the vaccinia DNA ligase is nonessential for viral DNA replication and growth.
However, a virus DNA ligase-deficient mutant shows attenuated virulence in vivo and is
anomalously sensitive to DNA-damaging agents during infection, implying a role for the
enzyme in viral DNA repair.
T4 DNA ligases:
T4 DNA ligase is an enzyme encoded by bacteriophage T4. It also catalyzes the covalent
joining of two segments to one uninterrupted strand in a DNA duplex, provided that no
nucleotides are missing at the junction (repair reaction). For its catalytic activity the
enzyme requires the presence of ATP and Mg++. DNAs that lack the required phosphate
residues can be rendered capable of ligation by phosphorylation with T4 polynucleotide
kinase.

Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and
3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive
end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA
hybrids.
The catalytic activity of the enzyme requires the presence of ATP and Mg++. DNAs that
lack the required phosphate residues can be rendered capable of ligation by
phosphorylation with T4 polynucleotide kinase. The enzyme also catalyzes an addition
reaction of phosphate between pyrophosphate and ATP. The ligation and the repair
catalyzed reactions of T4 DNA ligases are illustrated in the following:
T4 DNA ligase is mainly used in joining DNAZ molecules with compatible cohesive
termini, or blunt ended double stranded DNA to one another or to synthetic linkers.
It catalyzes a joining reaction between DNA molecules involving the 3' - hydroxy and the
5' - phosphate termini.
In addition the enzyme catalyzes an exchange reaction of phosphate between
pyrophosphate and ATP.
Applications:
Cloning of restriction fragments
Joining linkers and adapters to blunt-ended DNA
VECTORS: GENERAL CHARACTERISTICS OF VECTORS
Vector is an agent that can carry a DNA fragment into a host cell in which it is capable
of replication. If it is used only for reproducing the DNA fragment, it is called a cloning
vector. If it is used for expression of foreign gene, it is called an expression vector.
Properties of a good vector:

It should be autonomously replicating i.e. it should have ori region.
It should contain at least one selectable marker e. g. gene for antibiotic resistance.
It should have unique restriction enzyme site (only one site for one RE) for different REs
to insert foreign DNA.
It should be preferably small in size for easy handling.
It should have relaxed control of replication so that multiple copies can be obtained.
Vectors are of different types depending on the host. These are as follows:
Bacterial vectors
Yeast vectors
Plant vectors
Animal vectors
BRIEF ACCOUNT OF NATURALLY OCCURRING PLASMIDS PROMOTERS, MCS, ORI
AND MAKER GENE-LAC Z.
Protein synthesis, which is responsible for trait characteristics, requires genes to
undergo two steps: 1) transcription, or production of a messenger RNA (mRNA); and 2)
translation of mRNA into a protein. A gene has three major regions: the promoter,
coding region, and terminator. The promoter acts as the regulator for the level of gene
expression i.e. when, where and how much of the gene product (protein) is produced.
The coding region contains the information for making mRNA, which in turn specifies
the protein to be produced; while the terminator indicates the end of the gene.
Promoters regulate level of gene expression by specifying how many mRNAs are
produced (transcribed) for a given gene. The DNA sequence of the promoter region
interacts with transcription factor proteins that serve to recruit the cellular machinery
needed to produce the RNA transcripts. Transcription is performed by the enzyme, RNA
polymerase. The resultant RNA transcript is processed into mRNA, and then translated
into protein. The number of mRNAs produced is a primary factor determining the
amount of protein synthesized, which plays a role in determining the level of gene
expression.

Factors that bind to promoters react to signals from the organism or/and the
surrounding environment. The source and type of signal determines the type of
promoters that are activated. In genetic engineering, there are three major types of
promoters used, depending on the level of gene expression and specificity required:
Constitutive promoters: These facilitate expression of the gene in all tissues
regardless of the surrounding environment and development stage of the organism.
Such promoters can turn on the gene in every living cell of the organism, all the time,
throughout the organism’s lifetime. These promoters can often be utilized across
species. Examples of constitutive promoters that are commonly used for plants include
Cauliflower mosaic virus (CaMV) 35S, opine promoters, plant ubiquitin (Ubi), rice actin
1 (Act-1) and maize alcohol dehydrogenase 1 (Adh-1). CaMV 35S is the most commonly
used constitutive promoter for high levels of gene expression in dicot plants. Maize Ubi
and rice Act-1 are the currently the most commonly used constitutive promoters for
monocots.
Tissue-specific or development-stage-specific promoters:
These facilitate expression of a gene in specific tissue(s) or at certain stages of
development while leaving the rest of the organism unmodified. In the case of plants,
such promoters might specifically influence expression of genes in the roots, fruits, or
seeds, or during the vegetative, flowering, or seed-setting stage. If the developer wants a
gene of interest to be expressed in more than one tissue type for example the root,
anthers and egg sac, then multiple tissue-specific promoters may have to be included in
the gene construct.
An example of a tissue-specific promoter is the phosphoenolpyruvate (PEP) carboxylase
promoter which induces gene expression only in cells that are actively involved in
photosynthesis. In plant genetic engineering, this promoter is used for traits desired in
the shoot, leaves and sometimes the stem. Expression of genes controlled by this
promoter is reduced later in the growing season as the plant approaches senescence.
Inducible promoters:
These are activated by exogenous (i.e., external) factors. Exogenous factors may be
abiotic such as heat, water, salinity, chemical, or biotic like pathogen or insect attack.
Promoters that react to abiotic factors are the most commonly used in plant genetic
engineering because these can easily be manipulated. Such promoters respond to

chemical compounds such as antibiotics, herbicides or changes in temperature or light.
Inducible promoters can also be tissue or development stage specific.
Promoters can be derived directly from naturally occurring genes, or may be
synthesized to combine regulatory sequences from different promoter regions. The
promoters interact with other regulatory sequences (enhancers or silencers) and
regulatory proteins (transcription factors) to influence the amount of gene
transcription/expression.
These are of two types:
Chemically-regulated promoters: including promoters whose transcriptional activity
is regulated by the presence or absence of alcohol, tetracycline, steroids, metal and
other compounds.
The activity of this class of promoters is modulated by chemical compounds that either
turn off or turn on gene transcription. As prerequisites, the chemicals influencing
promoter activity typically.
Should not be naturally present in the organism where expression of the transgene is
sought;
Should not be toxic;
Should affect only the expression of the gene of interest;
Should be easy to apply or removal; and
Should induce a clearly detectable expression pattern of either high or very low gene
expression.
Preferably, chemically-regulated promoters should be derived from organisms distant in
evolution to the organisms where its action is required. For example, promoters to be
used in plants are mostly derived from organisms such as yeast, E. coli, Drosophila or
mammals.
Alcohol-regulated: Syngenta has several patents and patent applications in Europe
and Australia directed to a transcriptional system containing the alcohol dehydrogenase
I (alcA) gene promoter and the transactivator protein AlcR. Different agricultural
alcohol-based formulations are used to control the expression of a gene of interest
linked to the alcA promoter.

Tetracycline-regulated: Yale University and BASF AG have several patents and
patent applications filed in the United States, Europe, Australia and Canada covering
aspects of tetracycline-responsive promoter systems, which can function either to
activate or repress gene expression system in the presence of tetracycline. Some of the
elements of the systems include a tetracycline repressor protein (TetR), a tetracycline
operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA), which is
the fusion of TetR and a herpes simplex virus protein 16 (VP16) activation sequence.
Steroid-regulated: Numerous patent and patent applications are directed to steroid-
responsive promoters for the modulation of gene expression in plant and animal cells.
Metal-regulated: Promoters derived from metallothionein (proteins that bind and
sequester metal ions) genes from yeast, mouse and human are the subject matter of
several United States patents granted to Genentech, University Patents Inc.
and The University of California (Berkeley). DNA constructs having metal-regulated
promoters and eukaryotic cells transformed with them are claimed.
Pathogenesis-related (PR) proteins are induced in plants in the presence of particular
exogenous chemicals in addition to being induced by pathogen infection. Salicylic acid,
ethylene and benzothiadiazole (BTH) are some of the inducers of PR proteins. Promoters
derived from Arabidopsis and maize PR genes are the subject matter of patents granted
to Novartis and Pioneer Hi-Bred in the United States, Australia and Europe.
Physically-regulated promoters: including promoters whose transcriptional activity is
regulated by the presence or absence of light and low or high temperatures.
Promoters induced by environmental factors such as water or salt stress, anaerobiosis,
temperature, illumination and wounding have potential for use in the development of
plants resistant to various stress conditions. These promoters contain regulatory
elements that respond to such environmental stimuli.
Temperature-induced promoters include cold- and heat-shock-induced promoters. In
many cases, these promoters are able to operate under normal temperature conditions,
which vary according to the organism, but when either cold or heat is applied, the
promoters maintain activity. In addition, expression can be enhanced by the application
of higher or lower temperature as compared to the normal temperature conditions. One
of the best studied eukaryotic heat-shock systems is the one found in Drosophila (fruit
fly).

Heat-inducible promoters
Mycogen Plant Sciences, The United States Department of Health and Human Services
and The General Hospital Corporation have granted patents and patent applications
that relate in general to DNA sequences of heat shock promoters and methods for
expressing a gene of interest under the control of such promoters. Some of the
inventions relate to the use of the heat shock promoters in transformed plants, while
others do not specify the organism to be transformed. For some properties of heat shock
promoters please also refer to the plant ubiquitin promoters.
Light-regulated promoters
The IP portfolio of Calgene Inc includes a United States patent that claims the use of
light responsive promoters in plant cells.
The other patents presented in this section relate to light-regulated promoters isolated
from genes of specific organisms. The University of Warwick in UK, Suntory LTD in
Japan and Mycogen Plant Sciences in the USA have filed patents on the use of
promoters whose expression is induced by light, such as a promoter isolated from
myxobacterium and promoters whose expression is inhibited by light exposure, such as
a promoter isolated from a pea gene.
The patents are classified according to whether the promoters are Light-
inducible or Light-repressible.
CONSTRUCTION OF PBR 322, PBR325, PUC18 AND 19 VECTORS
In early cloning experiments, the cloning vectors used were natural plasmids, such as
Col E1 and pSC101. While these plasmids are small and have single sites for the
common restriction endonucleases, they have limited genetic markers for selecting
transformants. For this reason, considerable effort was expended on constructing, in
vitro, superior cloning vectors. The best and most widely used of these early purpose-
built vectors is pBR322. Plasmid pBR322 contains the ApR and TcR genes of RSF2124
and pSC101, respectively, combined with replication elements of pMB1, a Col E1-like
plasmid. The origins of pBR322 and its progenitor, pBR313, are shown in Fig. and
details of its construction can be found in the papers of Bolivar et al. (1977a,b).
The origins of plasmid pBR322.

A.
B. The boundaries between the pSC101, pMB1 and RSF2124-derived material.
The numbers indicate the positions of the junctions in base pairs from the unique
EcoRI site.
The molecular origins of plasmid pBR322. R7268 was isolated in London in 1963
and later renamed R1. 1, A variant, R1drd19, which was derepressed for mating
transfer, was isolated. 2, The ApR transposon, Tn3, from this plasmid was
transposed on to pMB1 to form pMB3. 3, This plasmid was reduced in size by
EcoRI* rearrangement to form a tiny plasmid, pMB8, which carries only colicin
immunity. 4, EcoRI* fragments from pSC101 were combined with pMB8 opened at
its unique EcoRI site and the resulting chimeric molecule rearranged by EcoRI*
activity to generate pMB9. 5, In a separate event, the Tn3 of R1drd19 was
transposed to Col E1 to form pSF2124. 6, The Tn3 element was then transposed
to pMB9 to form pBR312. 7, EcoRI* rearrangement of pBR312 led to the
formation of pBR313, from which (8) two separate fragments were isolated and
ligated together to form pBR322. During this series of constructions, R1 and Col
E1 served only as carries for Tn3. (Reproduced by courtesy of Dr G. Sutcliffe and
Cold Spring Harbor Laboratory.)

Plasmid pBR322 has been completely sequenced. The original published sequence
(Sutcliffe 1979) was 4362 bp long. Position O of the sequence was arbitrarily set
between the A and T residues of the EcoRI recognition sequence (GAATTC). The
sequence was revised by the inclusion of an additional CG base pair at position 526,
thus increasing the size of the plasmid to 4363 bp (Backman & Boyer 1983, Peden
1983). More recently, Watson (1988) has revised the size yet again, this time to 4361
bp, by eliminating base pairs at coordinates 1893 and 1915. The most useful aspect of
the DNA sequence is that it totally characterizes pBR322 in terms of its restriction sites,
such that the exact length of every fragment can be calculated. These fragments can
serve as DNA markers for sizing any other DNA fragment in the range of several base
pairs up to the entire length of the plasmid.
There are over 40 enzymes with unique cleavage sites on the pBR322 genome.
The target sites of 11 of these enzymes lie within the TcR gene, and there are sites for a
further two (ClaI and HindIII) within the promoter of that gene. There are unique sites
for six enzymes within the ApR gene. Thus, cloning in pBR322 with the aid of any one
of those 19 enzymes will result in insertional inactivation of either the ApR or the TcR
markers. However, cloning in the other unique sites does not permit the easy selection
of recombinants, because neither of the antibiotic resistance determinants is
inactivated.
Following manipulation in vitro, E. coli cells transformed with plasmids with inserts in
the TcR gene can be distinguished from those cells transformed with recircularized
vector. The former are ApR and TcS, whereas the latter are both ApR and TcR. In
practice, transformants are selected on the basis of their Ap resistance and then
replica-plated on to Tc-containing media to identify those that are TcS.
Cells transformed with pBR322 derivatives carrying inserts in the ApR gene can be
identified more readily (Boyko & Ganschow 1982). Detection is based upon the ability of
the β-lactamase produced by ApR cells to convert penicillin to penicilloic acid, which in
turn binds iodine. Transformants are selected on rich medium containing soluble
starch and Tc. When colonized plates are flooded with an indicator solution of iodine
and penicillin, β-lactamase-producing (ApR) colonies clear the indicator solution
whereas ApS colonies do not.

The PstI site in the ApR gene is particularly useful, because the 3′ tetranucleotide
extensions formed on digestion are ideal substrates for terminal transferase. Thus this
site is excellent for cloning by the homopolymer tailing method.
If oligo(dG.dC) tailing is used, the PstI site is regenerated and the insert may be cut out
with that enzyme. Plasmid pBR322 has been a widely used cloning vehicle. In addition,

it has been widely used as a model system for the study of prokaryotic transcription
and translation, as well as investigation of the effects of topological changes on DNA
conformation.
The popularity of pBR322 is a direct result of the availability of an extensive body of
information on its structure and function. This in turn is increased with each new
study. The reader wishing more detail on the structural features, transcriptional
signals, replication, amplification, stability and conjugal mobility of pBR322 should
consult the review of Balbás et al. (1986).
Example of the use of plasmid pBR322 as a vector: isolation of DNA fragments
which carry promoters
Cloning into the HindIII site of pBR322 generally results in loss of tetracycline
resistance. However, in some recombinants, TcR is retained or even increased. This is
because the HindIII site lies within the promoter rather than the coding sequence.
Thus whether or not insertional inactivation occurs depends on whether the cloned
DNA carries a promoter-like sequence able to initiate transcription of the TcR gene.
Widera et al. (1978) have used this technique to search for promoter-containing
fragments.
Four structural domains can be recognized within E. coli promoters. These are:
• Position 1, the purine initiation nucleotide from which RNA synthesis begins;
• Position −6 to −12, the Pribnow box;
• The region around base pair −35;
• The sequence between base pairs −12 and −35.
Although the HindIII site lies within the Pribnow box (Rodriguez et al. 1979) the box is
re-created on insertion of a foreign DNA fragment. Thus when insertional inactivation
occurs it must be the region from −13 to −40 which is modified.
Restriction map of plasmid pBR322 showing the location and direction of transcription
of the ampicillin (Ap) and tetracycline (Tc) resistance loci, the origin of replication (ori)
and the Col E1-derived Rop gene. The map shows the restriction sites of those enzymes
that cut the molecule once or twice. The unique sites are shown in bold type. The
coordinates refer to the position of the 5′ base in each recognition sequence with the
first T in the EcoRI site being designated as nucleotide number 1. The exact positions of

the loci are: Tc, 86–1268; Ap, 4084–3296; Rop, 1918–2105 and the origin of replication,
2535.
Much of the early work on the improvement of pBR322 centred on the insertion of
additional unique restriction sites and selectable markers, E.g. pBR325 encodes
chloramphenicol resistance in addition to ampicillin and tetracycline resistance and has
a unique EcoRI site in the CmR gene. Initially, each new vector was constructed in a
series of steps analogous to those used in the generation of pBR322 itself.
Then the construction of improved vectors was simplified (Vieira & Messing 1982, 1987,
Yanisch-Perron et al. 1985) by the use of polylinkers or multiple cloning sites (MCS), as
exemplified by the pUC vectors.
Over the years, numerous different derivatives of pBR322 have been constructed, many
to fulfil special-purpose cloning needs. A compilation of the properties of some of these
plasmids has been provided by Balbás et al. (1986).
An MCS is a short DNA sequence, 2.8 kb in the case of pUC19, carrying sites for many
different restriction endonucleases. An MCS increases the number of potential cloning
strategies available by extending the range of enzymes that can be used to generate a
restriction fragment suitable for cloning. By combining them within an MCS, the sites
are made contiguous, so that any two sites within it can be cleaved simultaneously
without excising vector sequences.
Improved vectors derived from pBR322
The pUC vectors also incorporate a DNA sequence that permits rapid visual detection of
an insert. The MCS is inserted into the lacZ′ sequence, which encodes the promoter
and the α-peptide of β- galactosidase. The insertion of the MCS into the lacZ′ fragment
does not affect the ability of the α-peptide to mediate complementation, but cloning
DNA fragments into the MCS does. Therefore, recombinants can be detected by
blue/white screening on growth medium containing Xgal.
The usual site for insertion of the MCS is between the iniator ATG codon and codon 7, a
region that encodes a functionally non-essential part of the α-complementation peptide.
Recently, Slilaty and Lebel (1998) have reported that blue/white colour selection can be
variable. They have found that reliable inactivation of complementation occurs only
when the insert is made between codons 11 and 36.

Cloning foreign DNA using the PstI site of pBR322.
Cut both the plasmid and the insert (yellow) with PstI, and then join them through
these sticky ends with DNA ligase.
Next, transform bacteria with the recombinant DNA and screen for tetracycline-
resistant, ampicillin-sensitive cells.
The recombinant plasmid no longer confers ampicillin resistance because the foreign
DNA interrupts that resistance gene (blue).
CONSTRUCTION OF EXPRESSION VECTORS
Expression vectors are required if one wants to prepare RNA probes from the cloned
gene or to purify large amounts of the gene product. In either case, transcription of the
cloned gene is required. Although it is possible to have the cloned gene under the
control of its own promoter, it is more usual to utilize a promoter specific to the vector.
Such vector-carried promoters have been optimized for binding of the E.coli RNA
polymerase and many of them can be regulated easily by changes in the growth
conditions of the host cell.

E. coli RNA polymerase is a multi-subunit enzyme. The core enzyme consists of two
identical α subunits and one each of the β and β′ subunits. The core enzyme is not
active unless an additional subunit, the σ factor, is present. RNA polymerase
recognizes different types of promoters depending on which type of σ factor is attached.
The most common promoters are those recognized by the RNA polymerase with σ70. A
large number of σ70 promoters from E. coli have been analysed and a compilation of
over 300 of them can be found in Lisser and Margalit (1993). A comparison of these
promoters has led to the formulation of a consensus sequence
The base sequence −10 and −35 regions of four natural promoters, two hybrid
promoters and the consensus promoter.
If the transcription start point is assigned the position +1 then this consensus sequence
consists of the −35 region (5′-TTGACA-) and the −10 region, or Pribnow box (5′-TATAAT).
RNA polymerase must bind to both sequences to initiate transcription. The strength of a
promoter, i.e. how many RNA copies are synthesized per unit time per enzyme molecule,
depends on how close its sequence is to the consensus. While the −35 and −10 regions
are the sites of nearly all mutations affecting promoter strength, other bases flanking
these regions can affect promoter activity (Hawley & McClure 1983, Dueschle et al.
1986, Keilty & Rosenberg 1987). The distance between the −35 and −10 regions is also
important. In all cases examined, the promoter was weaker when the spacing was
increased or decreased from 17 bp. Upstream (UP) elements located 5′ of the −35
hexamer in certain bacterial promoters are A+T-rich sequences that increase
transcription by interacting with the α subunit of RNA polymerase. Gourse et al. (1998)
have identified UP sequences conferring increased activity to the rrn core promoter. The
best UP sequence was portable and increased heterologous protein expression from the
lac promoter by a factor of 100.

Once RNA polymerase has initiated transcription at a promoter, it will polymerize
ribonucleotides until it encounters a transcription-termination site in the DNA.
Bacterial DNA has two types of transcription-termination site: factor-independent and
factor-dependent. As their names imply, these types are distinguished by whether they
work with just RNA polymerase and DNA alone or need other factors before they can
terminate transcription. The factor-independent transcription terminators are easy to
recognize because they have similar sequences: an inverted repeat followed by a string
of A residues.
Structure of a factor-independent transcriptional terminator.
Transcription is terminated in the string of A residues, resulting in a string of U
residues at the 3′ end of the mRNA. The factordependent transcription terminators have
very little sequence in common with each other. Rather, termination involves interaction
with one of the three known E. coli termination factors, Rho (ρ), Tau (τ) and NusA. Most
expression vectors incorporate a factor-independent termination sequence downstream
from the site of insertion of the cloned gene.
Vectors for making RNA probes
Although single-stranded DNA can be used as a sequence probe in hybridization
experiments, RNA probes are preferred.

Method for preparing RNA probes from a cloned DNA molecule using a phage SP6
promoter and SP6 RNA polymerase.
The reasons for this are that the rate of hybridization and the stability are far greater for
RNA–DNA hybrids compared with DNA–DNA hybrids. To make an RNA probe, the
relevant gene sequence is cloned in a plasmid vector such that it is under the control of
a phage promoter. After purification, the plasmid is linearized with a suitable restriction
enzyme and then incubated with the phage RNA polymerase and the four
ribonucleoside triphosphates. No transcription terminator is required because the RNA
polymerase will fall off the end of the linearized plasmid. There are three reasons for
using a phage promoter. First, such promoters are very strong, enabling large amounts
of RNA to be made in vitro. Secondly, the phage promoter is not recognized by the E. coli
RNA polymerase and so no transcription will occur inside the cell. This minimizes any
selection of variant inserts. Thirdly, the RNA polymerases encoded by phages such as
SP6, T7 and T3 are much simpler molecules to handle than the E. coli enzyme, since
the active enzyme is a single polypeptide. If it is planned to probe RNA or single-
stranded
DNA sequences, then it are essential to prepare RNA probes corresponding to both
strands of the insert. One way of doing this is to have two different clones
corresponding to the two orientations of the insert.
An alternative method is to use a cloning vector in which the insert is placed between
two different, opposing phage promoters (e.g. T7/T3 or T7/SP6) that flank a multiple
cloning sequence.
Since each of the two promoters is recognized by a different RNA polymerase, the
direction of transcription is determined by which polymerase is used.
Structure and use of the LITMUS vectors for making RNA probes. (a) Structure of
the LITMUS vectors showing the orientation and restriction sites of the four
polylinkers.

A further improvement has been introduced by Evans et al. (1995). In their LITMUS
vectors, the polylinker regions are flanked by two modified T7 RNA polymerase
promoters. Each contains a unique restriction site (SpeI or AflII) that has been
engineered into the T7 promoter consensus sequence such that cleavage with the
corresponding endonuclease inactivates that promoter. Both promoters are active
despite the presence of engineered sites. Selective unidirectional transcription is
achieved by simply inactivating the other promoter by digestion with SpeI or AflII prior
to in vitro transcription.
Since efficient labelling of RNA probes demands that the template be linearized prior to
transcription, at a site downstream from the insert, cutting at the site within the
undesired promoter performs both functions in one step. Should the cloned insert
contain either an SpeI or an AflII site, the unwanted promoter can be inactivated by
cutting at one of the unique sites within the polylinker.
Method of using the LITMUS vectors to selectively synthesize RNA probes from
each strand of a cloned insert.

E.COLI PROMOTERS
LAC PROMOTERS
It is usually advantageous to keep a cloned gene repressed until it is time to express it.
One reason is that eukaryotic proteins produced in large quantities in bacteria can be
toxic. Even if these proteins are not actually toxic, they can build up to such great levels
that they interfere with bacterial growth. In either case, if the cloned gene were allowed
to remain turned on constantly, the bacteria bearing the gene would never grow to a
great enough concentration to produce meaningful quantities of protein product. The

solution is to keep the cloned gene turned off by placing it downstream of an inducible
promoter that can be turned off. The lac promoter is inducible to a certain extent,
presumably remaining off until stimulated by the synthetic inducer
isopropylthiogalactoside (IPTG). However, the repression caused by the lac repressor is
incomplete (leaky), and some expression of the cloned gene will be observed even in the
absence of inducer. One way around this problem is to express a gene in a plasmid or
phagemid that carries its own lacI (repressor) gene, as pBS does.
The pBluescript vector:
This plasmid is based on pBR322 and has that vector’s ampicillin resistance gene
(green) and origin of replication (purple). In addition, it has the phage f1 origin of
replication (orange). Thus, if the cell is infected by an f1 helper phage to provide the
replication machinery, single-stranded copies of the vector can be packaged into
progeny phage particles. The multiple cloning site (MCS, red) contains 21 unique
restriction sites situated between two phage RNA polymerase promoters ( T7 and T3).
Thus, any DNA insert can be transcribed in vitro to yield an RNA copy of either strand,
depending on which phage RNA polymerase is provided. The MCS is embedded in an E.
coli lacZ′ gene (blue), so the uncut plasmid will produce the β-galactosidase N-terminal
fragment when an inducer such as isopropylthiogalactoside (IPTG) is added to
counteract the repressor made by the lacI gene (yellow). Thus, clones bearing the uncut
vector will turn blue when the indicator X-gal is added. By contrast, clones bearing
recombinant plasmids with inserts in the MCS will have an interrupted lacZ′ gene, so no
functional β-galactosidase is made. Thus, these clones remain white. The excess

repressor produced by such a vector keeps the cloned gene turned off until it is time to
induce it with IPTG.
TRP PROMOTERS
The main function of an expression vector is to yield the product of a gene—usually, the
more products the better. Therefore, expression vectors are ordinarily equipped with
very strong promoters; the rationale is that the more mRNA that is produced, the more
protein product will be made. One such strong promoter is the trp (tryptophan operon)
promoter. It forms the basis for several expression vectors, including ptrpL1.
Two uses of the ptrpL 1 expression vector:
The vector contains a ClaI cloning site, preceded by a Shine–Dalgarno ribosome binding
site (SD) and the trp operator–promoter region (trpO,P). Transcription occurs in a
counterclockwise direction as shown by the arrow (top). The vector can be used as a
traditional expression vector (left) simply by inserting a foreign coding region (X, green)
into the unique ClaI site. Alternatively (right), the trp control region (purple) can be cut

out with ClaI and HindIII and inserted into another plasmid bearing the coding region
(Y, yellow) to be expressed.
It has the trp promoter/operator region, followed by a ribosome binding site, and can be
used directly as an expression vector by inserting a foreign gene into the ClaI site.
Alternatively, the trp control region can be made “portable” by cutting it out with ClaI
and HindIII and inserting it in front of a gene to be expressed in another vector.
LAMBDA PL PROMOTERS
Another strategy is to use a tightly controlled promoter such as the λ phage promoter
PL. Expression vectors with this promoter–operator system are cloned into host cells
bearing a temperature-sensitive λ repressor gene (c1857). As long as the temperature of
these cells is kept relatively low (32° C), the repressor functions, and no expression
takes place. However, when the temperature is raised to the nonpermissive level (42° C),
the temperaturesensitive repressors can no longer function and the cloned gene is
derepressed.
A very popular method of ensuring tight control, as well as high-level induced
expression, is to place the gene to be expressed in a plasmid under control of a T7
phage promoter. Then this plasmid is placed in a cell that contains a tightly regulated
gene for T7 RNA polymerase. For example, the T7 RNA polymerase gene may be under
control of a modified lac promoter in a cell that also carries the gene for the lac
repressor. Thus, the T7 polymerase gene is strongly repressed unless the lac inducer is
present. As long as no T7 polymerase is present, transcription of the gene of interest
cannot take place because the T7 promoter has an absolute requirement for its own
polymerase. But as soon as a lac inducer is added, the cell begins to make T7
polymerase, which transcribes the gene of interest. And because many molecules of T7
polymerase are made, the gene is turned on to a very high level and abundant amounts
of protein product are made.
HYBRID TAC PROMOTERS
When maximizing gene expression it is not enough to select the strongest promoter
possible: the effects of overexpression on the host cell also need to be considered. Many
gene products can be toxic to the host cell even when synthesized in small amounts.
Examples include surface structural proteins (Beck & Bremer 1980), proteins, such as
the PolA gene product, that regulate basic cellular metabolism (Murray & Kelley 1979),

the cystic fibrosis transmembrane conductance regulator (Gregory et al. 1990) and
lentivirus envelope sequences (Cunningham et al. 1993). If such cloned genes are
allowed to be expressed there will be a rapid selection for mutants that no longer
synthesize the toxic protein.
Even when overexpression of a protein is not toxic to the host cell, high-level synthesis
exerts a metabolic drain on the cell. This leads to slower growth and hence in culture
there is selection for variants with lower or no expression of the cloned gene because
these will grow faster.
To minimize the problems associated with high-level expression, it is usual to use a
vector in which the cloned gene is under the control of a regulated promoter.
Many different vectors have been constructed for regulated expression of gene inserts
but most of those in current use contain one of the following controllable promoters: λ
PL, T7, trc (tac) or BAD.
Table shows the different levels of expression that can be achieved when the gene for
chloramphenicol transacetylase (CAT) is placed under the control of three of these
promoters.
The trc and tac promoters are hybrid promoters derived from the lac and trp promoters
(Brosius 1984). They are stronger than either of the two parental promoters because
their sequences are more like the consensus sequence. Like lac, the trc and tac
promoters are inducibile by lactose and isopropyl-β-d-thiogalactoside (IPTG). Vectors
using these promoters also carry the lacO operator and the lacI gene, which encodes the
repressor.
The pET vectors are a family of expression vectors that utilize phage T7 promoters to
regulate synthesis of cloned gene products (Studier et al. 1990). The general strategy for
using a pET vector is shown in Fig

Strategy for regulating the expression of genes cloned into a pET vector.
The gene for T7 RNA polymerase (gene 1) is inserted into the chromosome of E.
coli and transcribed from the lac promoter; therefore, it will be expressed only if
the inducer
IPTG is added. The T7 RNA polymerase will then transcribe the gene cloned into
the pET vector. If the protein product of the cloned gene is toxic, it may be
necessary to further reduce the transcription of the cloned gene before induction.
The T7 lysozyme encoded by a compatible plasmid, pLysS, will bind to any
residual T7 RNA polymerase made in the absence of induction and inactivate it.
Also, the presence of lac operators between the T7 promoter and the cloned gene
will further reduce transcription of the cloned gene in the absence of the inducer
IPTG.
To provide a source of phage-T7 RNA polymerase, E. coli strains that contain gene 1 of
the phage have been constructed. This gene is cloned downstream of the lac promoter,
in the chromosome, so that the phage polymerase will only be synthesized following
IPTG induction. The newly synthesized T7 RNA polymerase will then transcribe the
foreign gene in the pET plasmid. If the protein product of the cloned gene is toxic, it is
possible to minimize the uninduced level of T7 RNA polymerase. First, a plasmid
compatible with pET vectors is selected and the T7 lysS gene is cloned in it. When

introduced into a host cell carrying a pET plasmid, the lysS gene will bind any residual
T7 RNA polymerase (Studier 1991, Zhang & Studier 1997). Also, if a lac operator is
placed between the T7 promoter and the cloned gene, this will further reduce
transcription of the insert in the absence of IPTG (Dubendorff & Studier 1991).
Improvements in the yield of heterologous proteins can sometimes be achieved by use of
selected host cells (Miroux & Walker 1996).
The λ PL promoter system combines very tight transcriptional control with high levels of
gene expression. This is achieved by putting the cloned gene under the control of the PL
promoter carried on a vector, while the PL promoter is controlled by a cI repressor gene
in the E. coli host. This cI gene is itself under the control of the tryptophan (trp)
promoter,
In the absence of exogenous tryptophan, the cI gene is transcribed and the cI repressor
binds to the PL promoter, preventing expression of the cloned gene. Upon addition of
tryptophan, the trp repressor binds to the cI gene, preventing synthesis of the cI
repressor. In the absence of cI repressor, there is a high level of expression from the
very strong PL promoter. Many of the vectors designed for high-level expression also
contain translation-initiation signals optimized for E. coli expression.

RIBOSOME BINDING SITE
Translation of mRNA into protein is a complex process which involves interaction of the
messenger with ribosomes. For translation to take place the mRNA must carry a
ribosome binding site (rbs) in front of the gene to be translated. After binding, the
ribosome moves along the mRNA and initiates protein synthesis at the first AUG codon
it encounters and continues until it encounters a stop codon (UAA, UAG or UGA). If the
cloned gene lacks a ribosome binding site, it is necessary to use a vector in which the
gene can be inserted downstream from both a promoter and an rbs.
CODON SELECTION
The hallmark of bacterial adaptation to novel environments is physiological
differentiation, whereby evolved organisms interact with their environments differently
than did their ancestors. Such physiological differentiation often involves
change in biochemical activities as the result of gene gain, gene loss, or the occurrence
of mutations that change the biochemical activities of existing gene products. These
adaptive shifts can be readily identiﬁed as changes in gene inventory (Ochman et al.
2000; Hacker and Carniel 2001) or as sites showing evidence of positive selection for
change (Nielsen and Yang 1998; Suzuki and Gojobori 1999). However, exploration of the
novel ecological niches afforded by these changes may also demand expression changes
among genes not involved in qualitative physiological adaptations. For example,
changes in the abundance of a familiar nutrient will result in a concomitant change in
the demand for the enzymes to metabolize that nutrient. Here, adaptation can occur

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