Download MB 206 Microbial Biotechnology2

Cloning & Expression Vector
b
-
Reproductive
cloning
• The entire animal is produced from a single cell
by asexual reproduction. This would allow for the
creation of a human being who is genetically
identical to another.
Therapeutic
cloning
• Broader use of the term “cloning.” Does not
create a new genetically identical individual.
Research includes therapy for human
mitochondria disease and others that could
replace damaged or diseased tissues without the
risk of rejecting another’s tissue. Could create
new skin tissue for burn patients.
What is DNA cloning?
b
isolation and manipulation of
fragments of an organism’s genome
by replicating independently as part
an autonomous vector in another host
species.
b
DNA fragment in vector will form
recombinant DNA.
Basic steps in gene cloning
DNA
Vector
isolation
restriction
ligation
insert
Recombinant DNA
Host cells
Transformation/amplification
Selection / identification of clones
Validation of clones –analyses
RE, Southern blot, PCR, DNA sequencing
Positive recombinant DNA
The cloning of DNA
in a plasmid
Cloning Considerations
b
Choosing a vector
• Plasmids limited to small molecules
• Bacteriophages (phages) are viruses that
infect bacteria
• Phages can carry DNA inserts up to
15,000 nucleotides long
Cloning Considerations
Choosing a host
• Bacteria very good, but limitations
• Bacteria lack ability to modify proteins and limited
size of insert
• Yeast (Saccharomyces cere isiae) excellent for
many applications
• Occasionally necessary to clone genes into
specific animal or plant hosts – more difficult but
possible
Hosts & Vectors
b
Host systems:
- Bacterium (E.coli)
- Yeast (Saccharomyces cerevisea)
- Insect cells
- Mammalian (Chinese Hamster Ovary cells)
b
Cloning vectors
- derived from natural replicons
- Capable of replicating and isolation from host.
- Contain a selectable marker to distinguish host cells
containing the vector from amongst those that do not (eg.
antibiotic resistancy or survival under certain growth
conditions.
Types of Vectors
Plasmid DNA
E. coli vectors, extra-chromosomal and circular
Bacteriophages
Phage l – clone large DNA fragments and incorporate
into host genome
Phage M13 – allows cloned DNA to be isolated in
single-stranded form
Cosmids
hybrids of plasmid-bacteriophage l
Artificial chromosomes
- Cloning of very large genomic fragments
- BACs (bacterial artificial chromosomes)
- YACs (yeast artificial chromosomes
• viruses that infect bacteria
• known dsDNA sequence of ~ 50 kb
• linear double-stranded molecule with
single-stranded complementary ends
• cohesive termini (cos region)
http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif
Desirable properties of λ phage:
• can accept large pieces of foreign DNA
• tremendous improvement over the years
• can be reconstituted in vitro
Bacteriophage l
l phage genome linear 48.5 kb
genome.
Each ends consists
of cos (cohesive) sites
– 12 bp cos ends
Cos ends allows
DNA circularization in
the cell
Central region of
genome are nonessential portions and
can be replaced by
foreign DNA (up to
23kb)
Phage particles
injects linear DNA
into the cell
DNA ligate to form
circle
Replicate to form
many new phage
particles which are
released by cell
lysis and cell
death
or DNA intergrate
to host genome by
site-specific
recombination
(lysogenic phase)
Lysis plaques of l phage on
E. coli bacteria.
bacteria lawn
plaques
l bacteriophage
Plaques: the clear areas within the lawn
where lysis and re-infection have prevented
the cells from growing.
• small circular dsDNA that autonomously replicates
apart from the chromosome of the host cell
• “molecular parasites”
• carry one or more genes some of which confer
resistance to certain antibiotics
• origin of replication (ORI) --- a region of DNA that
allows multiplication of the plasmid within the
host
• plasmid replication: stringent or relaxed
Desirable properties of plasmids:
 small size
 known DNA sequence
 high copy number
 a selectable marker
 a second selectable gene
 large number of unique restriction sites
http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif
MB 206 : Module 3 - D
Transferring of Genetic Materials
Bacterial
transformation
 Process by which bacterial cells take up naked
DNA molecules.
 If the foreign DNA has an origin of replication
recognized by the host cell DNA
polymerases, the bacteria will replicate the
foreign DNA along with their own DNA.
Generic
Plasmid
Polycloning Site:
Region rich in restriction sites
that make for convenient
insertion of foreign DNA.
Selectable Marker:
Antibiotic resistance genes
that facilitate the
identification of bacteria
which contain plasmids.
Bacterial
Replicon:
Origin of replication and cis-acting
control elements that regulate plasmid
copy number (1-700 copies/cell)
Transferring
Genetic Materials
Chemical
method
(use salt and heatshock) to promote
DNA uptake.
Method of transferring
genetic material to
recipient cells without
the need for
conjugation.
Electroporation
electric shock to
facilitate DNA
uptake.
Advantage vs Disadvantage
•Heat-Shock Transformation:
– Quick and dirty but not very efficient
•Electroporation:
– More tedious but also far more efficient.
b -Galactosidase Gene
The protein encoded by this gene turns cells
blue. Insertion of foreign DNA in the middle
of this gene screws up the protein so cells
appear white.
PCS
Ampr
Only bacteria which
contain this plasmid
can grow on agar
containing ampicillin.
All other bacteria die.
Bacteri
al
Replico
n
In the presence of selective
media and X-Gal only
transformed cells grow and
those without inserts turn
blue.
CLONING VECTORS
 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.
PLASMID VECTORS
 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.
Exogenous DNA can be
inserted into the bracketed
region .
SELECTIVE MARKER
 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
 Origin of replication is a
DNA segment
recognized by the cellular
DNA-replication
enzymes.
 Without replication
origin, DNA cannot be
replicated in the cell.
MULTIPLE CLONING SITE
 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
 Restriction mapping: determining the order of
restriction sites in a cloned fragment:
 Gel electrophoresis: separates DNA fragments
by molecular weight
 Southern Blot analysis: DNA is transferred
("blotted") to filter paper.Filter is exposed to a
DNA probe. Binds specifically to target DNA
immobilized on filter
 DNA sequencing: provides complete order of
bases in a DNA fragment
RECOMBINANT DNA
 R.E. are a useful tool for analysing Recombinant DNA
▪ checking the size of the insert
▪ checking the orientation of the insert
▪ determining pattern of restriction sites within insert
 Sometimes it is important to determine the orientation
of the DNA insert in relation to the vector sequence.
 This can be done simply by restriction digest using
enzyme(s) which cut the vector sequence near to the
insert and cut within the insert sequence
(asymmetrically).
APPLICATIONS
 Cloning DNA fragments
 Generating Libraries: essential step for
genome mapping
 Positional cloning – discovering disease genes
 Discovering genes from e.g. Protein sequence
MB206
CDNA LIBRARY
Angelia 09
32
Types of Libraries
Genomic Library
• whole genes w/ promoters & introns (Euk.), operons (bacteria),
DNA regulatory elements…
cDNA Library
• mRNA transcript only w/ 5’ & 3’ untranslated regions (UTRs), no
introns, tissue specific.
(5’UTR)
(3’UTR)
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Angelia 09
mRNA isolation, purification
Check the RNA integrity
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector