Genetic Manipulations

of Bacteria
Prof. Dr. Isa GKE
Gaziosmanpasa University Faculty of Engineering
Department of Bioengineering

constructed based on rRNA

sequences. Hyperthermophiles
represented with thick red lineages
(Stetter, 2006).

Genotype of an E. coli
strain

DH5

F/endA1 hsdR17 (rK mK+) glnV44 thi1 recA1 gyrA (NalR) relA1 (lacIZYAargF)U169 deoR (80dlac(lacZ)M15)

Taq DNA polymerase

A thermophilic bacteria called Thermus
aquaticus is the source of Taq DNA polymerase
used in PCR reactions.

Biotechnology
The use of living cells to make
products such as pharmaceuticals,
foods, and beverages
The use of organisms such as bacteria
to protect the environment
The use of DNA science for the
production of products, diagnostics,
and research

Recombination

Recombination
Insert a foreign gene into a host
Plasmid ( for example, exogenous DNA)
into the bacterial cell transformation
or transfection-organism referred to as
transgenic ( eukaryote ) or
recombinant( prokaryote)
Goal To produce many copies
( clones) of a particular gene
Reporter gene tags gene of interest
to identify the presence of a gene

Datsenko K A , and Wanner B L PNAS 2000;97:6640-6645

A simple gene disruption strategy.

Manipulating DNA
How are changes made to DNA?
Scientists use their knowledge of the
structure of DNA & its chemical
properties to study & change DNA
molecules
*This is called GENETIC ENGINEERING

Alternative names for genetic engineering:

Genetic Manipulation

Genetic Modification

Recombinant DNA Technology

Recombineering

Gene Splicing

Gene Cloning

Genetic Engineering : Making desired changes

in the DNA code of a living organism

Genetic Engineering breaks the species

barrier

Genetic engineering allows DNA from

different species to be joined together.

This often results in combinations of

DNA that would never be possible in
nature. For this reason genetic
engineering is not a natural process.
If DNA is transferred from one species
to another the organism that receives
the DNA is said to be transgenic.

Why is genetic engineering important?

Purify protein
Insulin
Growth factor
Interferon
Bacteria can be engineered to eat oil spills

Generate more copies of a particular gene:

amplify DNA
Research gene function and regulation

Pharmaceuticals

insulin for diabetics

factor VIII for males suffering from hemophilia A
factor IX for hemophilia B
human growth hormone
erythropoietin for treating anemia
three types of interferons
interleukins
tissue plasminogen activator for dissolving blood clots
angiostatin and endostatin for trials as anti-cancer drugs
parathyroid hormone
leptin
hepatitis B surface antigen (HBsAg) to vaccinate against the
hepatitis B virus

Tools Used in Genetic Engineering

Tools used in genetic engineering!!!

Source of DNA: Target (foreign) DNA
DNA taken from one organism to be
placed into the DNA of a second
organism.
A cloning vector: Special kind of DNA
that can accept foreign DNA and exactly
reproduce itself and the foreign DNA e.g.
Bacterial plasmid (loop of DNA found in
bacteria).

Tools Used in Genetic Engineering

Restriction Enzymes: Restriction
endonucleases
Special enzymes used to cut the DNA at
specific places.
different enzymes cut DNA at specific
base sequences known as a recognition
site. For example
i) One restriction enzyme will always cut
DNA at
the base sequence: GAATTC.
ii) Another restriction enzyme only cuts at
the
sequence: GATC
If DNA from two different organisms is cut with

Restriction
enzymes
DNA
1

DNA
2

Tools for Recombination

Restriction enzymes

Tools used in Genetic

Engineering
DNA Ligase: enzyme
which acts like a glue
sticking foreign DNA to
DNA of the cloning vector.
Will work better if DNA
from the two DNA sources
has been cut with the
same restriction enzyme
i.e. sticky ends of cut DNA
will be complementary to
each other.

Process of Genetic Engineering

Five steps involved in this process:
1. Isolation
2. Cutting
3. Insertion (Ligation)
4. Transformation
5. Expression

Process of Genetic Engineering

a) DNA extraction simple chemical
process to get DNA out of cell; cells
are opened & DNA is separated from
other cell parts
b) Cutting DNA restriction
enzymes are used to cut DNA at
specific sequences of nucleotides

Process of Genetic Engineering

Insertion:
means that target gene is placed into the
DNA of the plasmid or cloning vector.
cut plasmids are mixed with human DNA
sections allowing the cut ends to
combine.
Transformation
Expression

Process of Genetic Engineering

c) separating & analyzing DNA
Scientist use gel electrophoresis
=
DNA fragments are put at one end
of a gel electric current is applied to
gel and DNA molecules move

DNA can be inserted into cell by:

Transformation
Treat cells (E. coli and other
bacteria, yeast) to make competent
Soak E.coli in CaCl2, mix with DNA,
mild heat shock (42 C)

DNA can be inserted into cell by:

Electroporation
Cells with cell wall need to be
converted to protoplasts

Sources of the DNA that is

inserted into the vector?
Gene library
collection of clones that
contain every gene of
an organism
pieces of an entire
genome stored in
plasmids or phages
Synthesize DNA with a
DNA machine (synthetic
gene)

Recombinant DNA
The manipulation and combination
of DNA from two sources
Bacterial DNA + human gene for
insulin
Plant DNA + bacterial DNA Agrobacterium tumefaciens
Mouse DNA + human DNA =
transgenic

Vectors
Plasmids
Bacteriophages Viruses
Particles ( DNA coated
bullets)
Exogenous DNA

Plasmids vehicles for

cloning

Plasmids are naturally occurring

extrachromosomal DNA molecules.
Plasmids are circular, double-stranded
DNA.
Plasmids are the means by which
antibiotic resistance is often
transferred from one bacteria to
another.
Plasmids can be cleaved by restriction
enzymes, leaving sticky or blunt ends.
Artificial plasmids can be constructed
by linking new DNA fragments to the
sticky ends of plasmid.

Ampr
Ori

pBR322
4361bp

Tetr

LacZ
MCS

pUC18
Ampr

Ori

pET28 Expression Vector

pTOL Expression Vector

Ap

ori

T7 promotor

XbaI

XhoI

KpnI
MluI

BamHI

MHHHHHHSSN329-P421GGGSXXXXX (X) GSGT

pTOLE

pTOLT
pTOLX

BamHI StuI
XhoI
ApaI NdeI NotI
KpnI
MluI
ggt ggg gga tct gat gat gac gat aaa gga tcc agg cct gag ctc cgg gcc cca tat ggc ggc cgcggt acc tga tga acg cgt
G G G S D D D D K G S R P E L R A P Y G G R G T * *
T R
BamHI StuI
XhoI
ApaI NdeI NotI
KpnI
MluI
ggt ggg gga tct ctg gtt ccg cgc gga tcc agg cct gag ctc cgg gcc cca tat ggc ggc cgcggt acc tga tga acg cgt
G G G S L V P R G S R P E L R A P Y G G R G T * *
T R
BamHI StuI
XhoI
ApaI NdeI NotI
KpnI
MluI
ggt ggg gga tct att gaa gggt cgc gga tcc agg cct gag ctc cgg gcc cca tat ggc ggc cgcggt acc tga tga acg cgt
G G G S I E G R G S R P E L R A P Y G G R G T * *
T R

Characteristics of a Vector
Can replicate independently in the
host cell
Has restriction sites in the vectorPolylinker cloning region
Has a reporter gene that will
announce its presence in the host cell
Is a small size in comparison to the
host chromosome for ease of isolation

Recombinant DNA

Making Recombinant
DNA Gene Splicing

1) Isolate pieces of
DNA with desirable
gene.
2) Inserting DNA into
host plasmid which
is bacteria or a virus.
3) Cloning vector.
4 )Selecting clones
with desired genes.

Site Directed
Mutagenesis

Bacterial Systems

Advantages

Easy and rapid

culture (protein
production in 8
hours )
High yields (50500 mg/L)
Cheap medium
(simple medium
content )
Low-cost
Fermentor

Disadvantages

Not suitable for

High molecular
weight proteins
(>50 kD)
Glikolization or
signal peptide
removal
Toxic to the host
organisms for some
eukaryotic proteins
Disulphide (S-S)
rich proteins

Why E . coli
Well known Genome and 60 years
experience
Suitable strains for desired proteins
Simple and quick cloning
Simple and cheap madium and
culturing
Labeling with isotopes (C13, N15)
Not pathogen
Many vectors for desired protein

Expression of Proteins in E. coli

Target protein + Fusion prot

Full length protein:
Protein+GST

Target
protei
n

Full length protein:

Advantages
suitable for 30-50
kDa proteins
No need for
cleaving with
specific proteases
Ideal for
functional and
structural studies
Proteazlara daha
az

Disadvantages
Denatured
Proteinsinclusion body
Sometimes yield
is too low
Production of
Low molecular
weight proteins
are problem.
Too many steps

Target protein + Fusion protein

Advantages

High yield
Suitable for
Production of Low
molecular weight
proteins
Where to Localise?
Helpful to folding
and solubilisation

Disadvantages
Needs specific
proteases
Sensitive to
proteolitic
digestion

E. coli cells
Cell distruption

300 mM Imidazole

Ultracentrifugation(400000rpm)
Supernatant
6His- Fusion and E. coli proteins
NiNTA agarose

6His -Fusion

SDS PAGE
1 2 3 4 5 6 7 8 9 10

NiNTA- 6His-Fusion

66 kDa
45 kDa
36 kDa
29 kDa
24 kDa
20.1 kDa

E. coli proteins

SDS-PAGE of produced and purified TOLAIII-

ColNT fusion protein

6
61
49
36
25
19
13

SDS-PAGE of produced and purified BClXL

fusion protein

SDS-PAGE of produced and purified T.

aquaticus SSB protein

SDS-PAGE of produced
Colicin A

Proteina
zK

SDS-PAGE of produced
Proteinase K

SDS-PAGE of produced and partially

purified GFP

SDS-PAGE of purified MBP+BClxl

Thank you

Erhan PKN
Jeremy LAKEY
University

Hacettepe University
Newcastle