Prof. Dr.

Mohamed El-Awady
1- Definition

2- Tools of genetic engineering.

3- Principles of DNA cloning.

4-Applications of Genetic Engineering.

Modification of the DNA of an organism to
produce new genes with new characteristics
- Restriction enzymes (Biological scissors).
- DNA Ligation (biological glue).
- Cloning vectors.
 Restriction: restrict the host range of the virus

Endonucleases: cut at sites within the foreign DNA

 Restriction enzymes:
enzymes that cut DNA in specific places function:

 Breaks only palindrome sequences, i.e. those exhibiting two-

fold symmetry

 Important in DNA research, i.e. sequencing, hybridization

 Therestriction enzymes most used
in molecular biology labs cut
within their recognition sites and
generate one of three different
types of ends.
 5' overhangs: The enzyme cuts asymmetrically
within the recognition site such that a short single-
stranded segment extends from the 5' ends. Bam HI
cuts in this manner.
 3' overhangs: Again, we see asymmetrical cutting
within the recognition site, but the result is a single-
stranded overhang from the two 3' ends. KpnI cuts in
this manner.
 Blunts: Enzymes that cut at precisely opposite sites
in the two strands of DNA generate blunt ends
without overhangs. SmaI is an example of an
enzyme that generates blunt ends.
 Cloning vectors are DNA molecules that are used to
"transport" cloned sequences between biological hosts
and the test tube.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.
2. Contain a genetic marker (usually dominant) for
selection.
3. Unique restriction sites to facilitate cloning of insert
DNA.
4. Minimum amount of nonessential DNA to optimize
cloning.
TYPES OF CLONING VECTORS
 Different types of cloning vectors are used for
different types of cloning experiments.
 The vector is chosen according to the size
and type of DNA to be cloned
 Bacterial cells may
contain extra-
chromosomal DNA
called plasmids.
 Plasmids are usually
represented by
small, circular DNA.
 Some plasmids are
present in multiple
copies in the cell
 Plasmid vectors are ≈1.2–3kb
and contain:
 replication origin (ORI) sequence
 a gene that permits selection,
 Here the selective gene is ampr;
it encodes the enzyme b-
lactamase, which inactivates
ampicillin.
 Multiple cloning site, a DNA
segment with several unique
sites for restriction endo-
nucleases located next to each
other
 Selective marker is required for
maintenance of plasmid in the cell.
 Because of the presence of the
selective marker the plasmid
becomes useful for the cell.
 Under the selective conditions, only
cells that contain plasmids with
selectable marker can survive
 Genes that confer resistance to
various antibiotics are used.
 Genes that make cells resistant to
ampicillin, neomycin, or
chloramphenicol are used
 Origin of replication
is a DNA segment
recognized by the
cellular DNA-
replication enzymes.
 Without replication
origin, DNA cannot
be replicated in the
cell.
 Many cloning vectors contain a
multiple cloning site or
polylinker: a DNA segment with
several unique sites for
restriction endo- nucleases
located next to each other
 Restriction sites of the polylinker
are not present anywhere else in
the plasmid.
 Cutting plasmids with one of the
restriction enzymes that
recognize a site in the polylinker
does not disrupt any of the
essential features of the vector
 Gene to be cloned
can be introduced
into the cloning
vector at one of the
restriction sites
present in the
polylinker
 Plasmid vectors are used to
clone DNA ranging in size
from several base pairs to
several thousands of base
pairs (100bp -10kb).
 commercially available
ones, eg pGEM3,
pBlueScript, pUC18….
 small size (easy to manipulate and isolate)
 circular (more stable)
 replication independent of host cell
 several copies may be present (facilitates
replication)
 frequently have antibody resistance (detection
easy)
 Cannot accept large fragments
 Sizes range from 0- 10 kb
 Standard methods of transformation
are inefficient
 Purpose:
1. Clone large inserts of
DNA: size ~ 45 kb
 Features:
Cosmids are Plasmids with
one or two Lambda Cos
sites. Cosmids' (cos sites +
plasmid = cosmid)
 Presence of the Cos site
permits in vitro packaging
of cosmid DNA into
Lambda particles
 Thus, have some advantages of Lambda as
Cloning Vehicle:
 Strong selection for cloning of large inserts
 Infection process rather than transformation for
entry of chimeric DNA into E. coli host
 Maintain Cosmids as phage particles in solution
 But Cosmids are Plasmids:
Thus do NOT form plaques but rather cloning
proceeds via E. coli colony formation
Yeast artificial chromosomes (YACs) are genetically
engineered chromosomes derived from the DNA of the
yeast, Saccharomyces cerevisiae, which is then ligated
into a bacterial plasmid. By inserting large fragments of
DNA, from 100–1000 kb, the inserted sequences can be
cloned and physically mapped using a process called
chromosome walking.
 Purpose:
 Cloning vehicles that propogate in
eukaryotic cell hosts as eukaryotic
Chromosomes

 Clone very large inserts of DNA: 100 kb - 10

Mb

 Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule
with telomeric ends: Artificial Chromosome
 A bacterial artificial chromosome (BAC) is a
DNA construct, based on a functional fertility
plasmid (or F-plasmid), used for transforming
and cloning in bacteria, usually E. coli.
 DNA cloning is a technique for enormous amplification of
DNA sequences .
 Stable propagation of DNA sequences
 A single DNA molecule can be amplified allowing it to be:
▪ Studied - Sequenced
▪ Manipulated - Mutagenised or Engineered
▪ Expressed - Generation of Protein
 It can be achieved by two different approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
 Involves five steps:
1- Enzyme restriction digest of DNA sample.
2- Enzyme restriction digest of DNA plasmid vector.
3- Ligation of DNA sample products and plasmid vector.
4- Transformation with the ligation products.
5- Growth on agar plates with selection for antibiotic
resistance.
 Gene of interest is cut
out with RE
 Host plasmid is cut
with same RE
 Gene is inserted into
plasmid and ligated
with ligase
 New plasmid inserted
into bacterium
(transform)
 The process of transferring exogenous DNA
into cells is call “transformation”
 There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to
promote DNA entry into cells.
 A second method is called electroporation
based on a short pulse of electric charge to
facilitate DNA uptake.
 Blue colonies represent Ampicillin-resistant
bacteria that contain pVector and express a
functional alpha fragment from an intact LacZ
alpha coding sequence.

 White colonies represent Ampicillin-resistant

bacteria that contain pInsert and do not produce
LacZ alpha fragment
 Blue-white screening is a rapid and efficient technique for the identification of
recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme
occurring in E. coli, which cleaves lactose into glucose and galactose.

 The presence of lactose in the surrounding environment triggers the lacZ operon in
E. coli. The operon activity results in the production of β-galactoisdase enzyme
that metabolizes the lactose.

 The plasmid vectors used in cloning are manipulated in such a way that this α-
complementation process serves as a marker for recombination. A multiple cloning
site (MCS) is present within the lacZ sequence in the plasmid vector. This
sequence can be nicked by restriction enzymes to insert the foreign DNA.

 Principle For screening the clones containing recombinant DNA, a chromogenic

substrate known as X-gal is added to the agar plate. If β-galactosidase is produced,
X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously
dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-
indigo. The colonies formed by non-recombinant cells, therefore appear blue in
color while the recombinant ones appear white.

 Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-

white screening. IPTG is a non-metabolizable analog of galactose that induces
(inducer) the expression of lacZ gene.
 DNA recombination.
The DNA fragment to be cloned is inserted into
a vector.
 Transformation.
The recombinant DNA enters into the host cell
and proliferates.
 Selective amplification.
A specific antibiotic is added to kill E. coli
without any protection. The transformed E. coli
is protected by the antibiotic-resistance gene
4-Application of Genetic Engineering
in Plants

- In plant
- In animals
-In medicine and pharmacy
- Other applications
Non transgenic
plants

Transgenic
plants
Non transgenic Transgenic
plants plants
 Non transgenic
plants

Transgenic plants
Application of Genetic Engineering
in Animals
 Transgenic Cattle

Dairy cows carrying extra copies of two types of

casein genes produce 13% more milk protein
Not only will this make the milk more nutritious,
it would allow for less milk to make more cheese
 Transgenic Fish
Salmon/trout
Catfish
Can grow up to 6 times faster than wildtype fish
Most have extra copies of growth hormone (GH) gene

Transgenic
Wildtype
Application of Genetic Engineering
in Pharmacy
1997, Tracy the sheep, the first transgenic animal to
produce a recombinant protein drug in her milk
alpha-1-antitrypsin (AAT) treatment for
emphysema ‫ & إنتفاخ الرئة‬cystic fibrosis ‫التليف‬
 Human protein C Anti-thrombin
3 for Treatment of
thrombosis ‫التخثر‬.
 Human gene for alpha1-
antitrypsin for Treatment of
disease Alpha1-Antitrypsin
Deficiency ‫عوز ضد التربسين = إنتفاخ الرئة‬
 Expressing human α-lactalbumin
(the major whey protein in
human milk) in the milk of
transgenic cattle.
 High-protein milk for the cheese
industry.
Nexia Biotechnologies transferred the silk gene from Orb
spiders into goats
Each goat produces several grams of silk protein in her milk
The silk is extracted, dried to a white powder, and spun into
fibers
The fibers are stronger and more flexible than steel
Other applications of Genetic
Engineering
Transgene ->
Gene coding
for a growth
hormone
 Alba, the EGFP (enhanced GFP) bunny
Created in 2000 as a transgenic artwork

The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues
(26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to
ultraviolet range
GloFish, originally developed in Singapore as a way to
monitor water pollution
The normally black-and-silver zebrafish was turned
green or red by inserting various versions of the GFP
gene
 normal knockout

GDF8 (Myostatin) knockout mouse

Over twice the muscle mass of a wildtype mouse
 Transgenic Clones

Cloned transgenic cat containing red fluorescent protein

‫تمت بحمد هللا ‪.........‬‬