Genetic Engineering and Biotechnology
? Genetic engineering,Isolation,
manipulation and expression of genetic
materials.
? Gene cloning,Isolation and purification of
specific genes
? In vitro recombination=recombinant DNA
Gene Cloning Procedures
? Isolation and fragmentation of the source DNA
? Joining the DNA fragments to a cloning vector
with DNA ligase
? Incorporation into a host
? Detection and purification of the desired clone
? Production of large numbers of cells or
bacteriophage containing the desired clone for
isolation and study of the clone DNA
Plasmids as Cloning Vectors
Small,only 4361 BP,
Stable in E,coli,
High copy number (1000-3000
copies per cell)
Easily isolated in the
supercoiled form
Foreign DNA can be inserted in
good amount
Restriction sites are known
Single cleavage sites for several
restriction enzymes
Two antibiotic resistance
markers
Transformation easy
Plasmid pBR322
Gene cloning and Expression
using Plasmid pBR322
Bacteriophages as Cloning Vectors
Modified lambda phages as cloning vectors
Cosmids,plasmid vectors
containing foreign DNA
plus only the cos (cohesive
end) site from the lambda
genome
? Sizes of a cloning gene the
vectors can carry:
YAC>Cosmid>Lambda>Plasmids
Other Vectors
? Yeast artificial chromosomes (YAC)
YAC vectors are only about 10 KB,but can carry 200-800 KB
DNA sequences
YAC has been developed and used for the human genome project
Vectors for DNA Sequencing,Bacteriophage M13
? Phagemid,a hybrids
between a
filamentous phage,
like M13 and a
plasmid
X-gal,5-bromo-4-
chloro-3-indolyl-b-D-
galactopyranoside
Vectors without
cloned DNA
Vectors with cloned
DNA
Other specialized vectors
? Expression vectors,to obtain synthesis of
the protein coded for by the foreign gene
cloned into the vector.
? Secretion vectors,protein product is not
only expressed but secreted (excreted) from
the cell.
? Shuttle vectors,move DNA between
unrelated organisms,can replicate in two
different organisms
Hosts for cloning vectors
? Ideal hosts,rapid growth,capable of growth in
cheap culture medium,not harmful or pathogenic,
transformable by DNA,stable.
? Prokaryotic hosts,E,coli,Bacillus subtilis.
? Eukaryotic hosts,Saccharomyces cerevisiae.
? DNA virus SV40,a virus causing tumors in
primates,can be used as a cloning vector into
human culture lines.
? Retroviruses,vaccinia virus are useful too.
? Baculovirus,an insect DNA virus can be used to
transfer DNA to insect cell lines
Finding
the right
clones
(a) Detecting production
of protein by use of
specific antibody
(b) Detecting
recombinant clones by
colony hybridization
with radioactive
nucleic acid probe
Finding the right clones
? Cloning and
expression of
luciferase genes
from various
types of
bioluminescent
beetles into E,
coli.
Expression Vector
? An expression vector for fusions,This is a phagemid,
can be propagated either as a plasmid or as a phage
Synthetic DNA
? Automatically synthesized
? 30-35 BP
? Used as probes
? Used as primers for PCR
? Used for Site-directed
mutagenesis
PCR,amplifying DNA
Organization of the Human Genome
DNA Fingerprinting
VNTR,Variable number of tandem repeats
Cloning and
expression of
mammalian genes
in bacteria
? mRNA route
? Protein route
The Synthesis of
Complementary DNA
(cDNA)
Reverse Translation
In vitro and
Site-Directed
Mutagenesis
Cassette
mutagenesis
and gene
disruption
Practical Applications of Genetic Engineering
? Microbial fermentations,antibiotics,enzymes,proteins,
biopolymers (PHA) etc.
? Virus vaccines,expression of virus protein coats.
? Mammalian proteins,cloning and expression of human
proteins in bacteria.
? Transgenic plants and animals,improve growth rates and
yields,as well as new properties for new species.
? Environmental biotechnology,clone new genes for
degradation of synthesized compounds.
? Gene regulation and gene therapy,antisense RNA,
ribosymes,genetic engineering to treat genetic diseases
Production of mammalian products and vaccines
by genetically engineered microorganisms
Production of Mammalian Products,Hormones
? Insulin for treating diabetics
? Human growth hormone for treating
dwarfism
? Epidermal growth factor for stimulating
wound healing
? Bone growth factors for treating
osteoporosis
? Animal growth hormone for stimulating
growth of livestock animals
Virus Vaccines
Production of recombinant
vaccinia virus
Genetic Engineering
in Plant Agriculture
? Agrobacterium tumefaciens
(根瘤土壤杆菌） can cause
the plant disease crown gall
by transferring specific genes
to the plant.
? Plant genetic engineers have
used this natural
transformation system as a
vehicle for the introduction of
foreign DNA into plants.
? A,tumefaciens contains a
large plasmid called Ti
plasmid which can mobilize
DNA for transfer to the plants
Genetic Engineering in Animal
and Human Genetics
? Living embryo of
a transgenic fish
that is expressing
the gene
encoding the
green fluorescent
protein under
control of a
Xenopus
promoter
? Genetic engineering,Isolation,
manipulation and expression of genetic
materials.
? Gene cloning,Isolation and purification of
specific genes
? In vitro recombination=recombinant DNA
Gene Cloning Procedures
? Isolation and fragmentation of the source DNA
? Joining the DNA fragments to a cloning vector
with DNA ligase
? Incorporation into a host
? Detection and purification of the desired clone
? Production of large numbers of cells or
bacteriophage containing the desired clone for
isolation and study of the clone DNA
Plasmids as Cloning Vectors
Small,only 4361 BP,
Stable in E,coli,
High copy number (1000-3000
copies per cell)
Easily isolated in the
supercoiled form
Foreign DNA can be inserted in
good amount
Restriction sites are known
Single cleavage sites for several
restriction enzymes
Two antibiotic resistance
markers
Transformation easy
Plasmid pBR322
Gene cloning and Expression
using Plasmid pBR322
Bacteriophages as Cloning Vectors
Modified lambda phages as cloning vectors
Cosmids,plasmid vectors
containing foreign DNA
plus only the cos (cohesive
end) site from the lambda
genome
? Sizes of a cloning gene the
vectors can carry:
YAC>Cosmid>Lambda>Plasmids
Other Vectors
? Yeast artificial chromosomes (YAC)
YAC vectors are only about 10 KB,but can carry 200-800 KB
DNA sequences
YAC has been developed and used for the human genome project
Vectors for DNA Sequencing,Bacteriophage M13
? Phagemid,a hybrids
between a
filamentous phage,
like M13 and a
plasmid
X-gal,5-bromo-4-
chloro-3-indolyl-b-D-
galactopyranoside
Vectors without
cloned DNA
Vectors with cloned
DNA
Other specialized vectors
? Expression vectors,to obtain synthesis of
the protein coded for by the foreign gene
cloned into the vector.
? Secretion vectors,protein product is not
only expressed but secreted (excreted) from
the cell.
? Shuttle vectors,move DNA between
unrelated organisms,can replicate in two
different organisms
Hosts for cloning vectors
? Ideal hosts,rapid growth,capable of growth in
cheap culture medium,not harmful or pathogenic,
transformable by DNA,stable.
? Prokaryotic hosts,E,coli,Bacillus subtilis.
? Eukaryotic hosts,Saccharomyces cerevisiae.
? DNA virus SV40,a virus causing tumors in
primates,can be used as a cloning vector into
human culture lines.
? Retroviruses,vaccinia virus are useful too.
? Baculovirus,an insect DNA virus can be used to
transfer DNA to insect cell lines
Finding
the right
clones
(a) Detecting production
of protein by use of
specific antibody
(b) Detecting
recombinant clones by
colony hybridization
with radioactive
nucleic acid probe
Finding the right clones
? Cloning and
expression of
luciferase genes
from various
types of
bioluminescent
beetles into E,
coli.
Expression Vector
? An expression vector for fusions,This is a phagemid,
can be propagated either as a plasmid or as a phage
Synthetic DNA
? Automatically synthesized
? 30-35 BP
? Used as probes
? Used as primers for PCR
? Used for Site-directed
mutagenesis
PCR,amplifying DNA
Organization of the Human Genome
DNA Fingerprinting
VNTR,Variable number of tandem repeats
Cloning and
expression of
mammalian genes
in bacteria
? mRNA route
? Protein route
The Synthesis of
Complementary DNA
(cDNA)
Reverse Translation
In vitro and
Site-Directed
Mutagenesis
Cassette
mutagenesis
and gene
disruption
Practical Applications of Genetic Engineering
? Microbial fermentations,antibiotics,enzymes,proteins,
biopolymers (PHA) etc.
? Virus vaccines,expression of virus protein coats.
? Mammalian proteins,cloning and expression of human
proteins in bacteria.
? Transgenic plants and animals,improve growth rates and
yields,as well as new properties for new species.
? Environmental biotechnology,clone new genes for
degradation of synthesized compounds.
? Gene regulation and gene therapy,antisense RNA,
ribosymes,genetic engineering to treat genetic diseases
Production of mammalian products and vaccines
by genetically engineered microorganisms
Production of Mammalian Products,Hormones
? Insulin for treating diabetics
? Human growth hormone for treating
dwarfism
? Epidermal growth factor for stimulating
wound healing
? Bone growth factors for treating
osteoporosis
? Animal growth hormone for stimulating
growth of livestock animals
Virus Vaccines
Production of recombinant
vaccinia virus
Genetic Engineering
in Plant Agriculture
? Agrobacterium tumefaciens
(根瘤土壤杆菌） can cause
the plant disease crown gall
by transferring specific genes
to the plant.
? Plant genetic engineers have
used this natural
transformation system as a
vehicle for the introduction of
foreign DNA into plants.
? A,tumefaciens contains a
large plasmid called Ti
plasmid which can mobilize
DNA for transfer to the plants
Genetic Engineering in Animal
and Human Genetics
? Living embryo of
a transgenic fish
that is expressing
the gene
encoding the
green fluorescent
protein under
control of a
Xenopus
promoter
