GENE CLONING AND RECOMBINANT DNA

TECHNOLOGY
 What is recombinant DNA?
 DNA from 2 different sources (often from 2 different
species) are combined together in vitro.
 Recombinant DNA forms the basis of cloning.

 Why is this useful?

 Allows scientists to manipulate organisms (e.g. E.
coli) to make useful products known as recombinant
proteins.
 Can be used to modify the phenotype of an organism.
These are called genetically modified organisms
(GMO).
 Used in biotechnology, medicine and research.
Generating recombinant DNA
HOW TO CLONE A GENE!
THE PRINCIPE OF CLONING
The cloning toolkit
Step 1 in gene cloning

 Vector DNA acts as a carrier for the DNA segment to be

cloned
 When a vector is introduced into a living cell, it can replicate
making many copies
 Common vectors are plasmid or viral
 Also need the gene of interest from chromosomal DNA
 Plasmid DNA
 Plasmids are found in bacterial cells.
 Small circular DNA molecules that replicate separately
from the bacterial chromosome.
 Plasmids as a tool for cloning
 Origin of replication
 Gene encoding a selectable marker
 Promoter
 Multiple cloning site
 A typical bacterial cloning vector

Antibiotic resistance
gene
Multiple cloning
site

Bacterial
promoter

Origin of replication
Preparing the DNA for cloning
 Restriction enzymes and cloning
 Enzymes that cut the DNA at a limited number of specific
locations
 Discovered in the late 1960s by biologists doing basic
research on bacteria
 In bacterial these protect the cell by cutting up DNA from other
organisms such as phages

 Hundreds of different restriction enzymes have been

identified
 Each enzyme recognises and cuts at a very specific sequence
of DNA
RESTRICTION ENZYMES AND CLONING
RESTRICTION ENZYMES AND CLONING
RESTRICTION ENZYMES AND CLONING
Inserting your gene of interest into the cloning vector
Ligating your Gene of Interest into the Cloning
Vector
DNA LIGASE
Making complementary DNA (cDNA) from
eukaryotic genes
BACTERIAL TRANSFORMATION
 Bacterial transformation
 Goal for recombinant vector to be taken up by bacteria
 Some will take up a single plasmid
 Most cells fail to take up a plasmid
 Vector carries a selectable marker
 Presence of antibiotics selects for cells expressing ampR
gene – contains plasmid
 ampR gene codes for b-lactamase that degrades
ampicillin, which normally kills bacteria
 Bacterial transformation
 After treatment, only cells with the plasmid will grow on
plates treated with ampicillin to eliminate recircularized
vectors from further examination, lacZ gene part of vector
 Insertion of chromosomal DNA disrupts lacZ gene
 lacZ codes for b-galactosidase which cleaves colorless X-
Gal into a blue dye
 Recircularized plasmids will form blue colonies
 Recombinant vectors will form white colonies
BACTERIAL TRANSFORMATION
COLLECTING THE RECOMBINANT PRODUCT
https://www.youtube.com/watch
?v=nfC689ElUVk
Applied recombinant DNA technology
RECOMBINANT PROTEIN
PRODUCTS
RECOMBINANT DNA TECHNOLOGY HAS BEEN
USED FOR MANY APPLICATIONS
 Insulin
 made by recombinant bacteria
 Prior to 1982, insulin isolated from cattle
 Some people developed allergies and had to use cadaver
insulin

 Insulin composed of 2 polypeptides – A and B

 A and B coding sequence inserted into E.coli
 Fusion proteins extracted and β-galactosidase removed
 Purified A and B chain mixed to form functional protein
INSULIN PRODUCTION
Applied recombinant DNA technology
CREATING GENETICALLY
MODIFIED PLANTS
 Why genetic modification?
 The world´s population is expanding (some statistic on
population expansion).
 To feed this growing population we need to find ways to
increase food production/nutrition from the land we have

 Desirable traits for plants

 Drought and stress tolerance
 Disease resistance
 Improved nutritional value
AGROBACTERIAM IS A USEFUL TOOL FOR
PLANT TRANSFORMATION
 Agrobacterium tumefaciens naturally infects plant cells and
causes tumors

© Copyright Malcolm Storey 2011-2115

 Contains Ti plasmid that integrates into host chromosome

 Codes for plant growth hormones that form crown gall tumor
GENERATING CLONING VECTORS FOR PLANT
TRANSFORMATION
GENE DELIVERY IN PLANTS
GENE DELIVERY IN PLANTS

Protoplast
Vector mediated
transformation
gene delivery via
Agrobacterium

Gene gun
delivery
PLANT REGENRATION
Tissue regeneration

 Plants are recovered on media containing all of the

nutrients required for plant growth (a carbon source,
vitamins, macro and micro nutrients, selection)

 Plants are growth and then tested for the presence of

the recombinant gene by PCR
Applied recombinant DNA technology
GENETICALLY MODIFIED
PLANTS
 Direct transformation of rice genotypes with
agronomically useful genes
Gene Trait Reference
psy, crt1, lyc Provitamin A biosynthesis Datta et al. (2003)
ferritin Iron Storage Vasconcelos et al. (2003)
FRO2 Resistance to insect pests Datta et al.
(unpublished)
Xa21 Bacterial leaf blight resistance Balchandrin et al.
(2003),
Narayanan et al. (2002,
2004)
Chitinase Sheath blight tolerance Baisakh et al. (2001)
Datta et al. (2001)
enod12 Early nodulation Reddy et al. (2001)
PEPC Increased photosynthesis Dtta et al. unpublished
glgC Increased starch biosynthesis Datta et al. unpublished
Adapted from table in Altpeter et al.
 Bacterial blight caused by Xanthomonas oryzae pv. oryzae is one of
the most destructive diseases of rice – causes losses of up to 50% in
areas of Asia
 Chemical control is not good at controlling the disease
 Xa21 is a bacterial blight resistance gene showing resistance to all
known strains of Xanthamonas in India and the Phillipines
 Transformed indica rice line IR72 with Xa21
 Xa21 is a bacterial blight
resistance gene showing
resistance to all known strains of
Xanthamonas in India and the
Phillipines.
 This gene was taken from a rice
plant that is not commercially
grown and cloned into a non-
resistant rice plant that is used
agriculturally.

T = transgenic
C = untransformed control
Tu et al. (1998) Theor appl genet
Xa21 in field trials

Genetically modified rice carrying Xa21 gene were tested

in the field and compared with wildtype rice plants

GM Rice Wildtype rice

Results of field tests were impressive - A

success story!
Case study: Glyphosate resistant plants

 Glyphosate = broad spectrum herbicide

 Inhibits enzymatic step in production of aromatic amino acid
pathway in plants and bacteria
 A “safe” herbicide due to natural degradation in soil to
harmless natural products
 Non-selective
 Transgenic crops resistant to Glyphosate would be very
useful!
 Concerns that herbicide resistance generated through
nuclear transformation could escape through pollen
dispersal into weeds – Super resistant weeds……
 Cloned the EPSPS gene from petunia, conferring
resistance to glyphosate into chloroplast expression
vector

•Shoots of tobacco were

transformed and regenerated
•Selected for spec/strep resistance
•Confirmed chloroplast gene
insertion by PCR
•Seeds collected from 1st self cross
were grown and tested:

Daniell et al. (1998) Nature

Biotechnology
Gene therapy using a retroviral vector