Download Vectors

Vectors
Vectors
1.
2.
3.
If a fragment of DNA is ligated into an appropriate
vector, it can be inserted into cells which will then
make many copies of it
Vectors are typically plasmids or viruses that have been
engineered to both accept DNA insertions and
reproduce inside cells
Cloning is the process of inserting DNA encoding a
gene of interest into a vector, then establishing it as a
stable part of a cell line.
pUC 18
A Typical Plasmid
Ampr
Gene
aagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattc
HindIII SphI PstI SalI XbaI BamHI XmaI KpnI SstI EcoRI
AccI
SmaI
BanII
HincII
BspMI
2,686 bp
Origin
of Replication
Lac Z
Gene
Multiple Cloning
Site
pUC 18 Sequence
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcaggg
cgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgt
aaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaaggg
ggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgcatgcctgcaggtcgactctaga
ggatccccgggtaccgagctcgaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtg
taaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggcca
acgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaa
ggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctgg
cgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctg
gaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaaagctcacgctgtaggtatct
cagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa
gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca
ctagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt
tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggatttt
ggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaa
tcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgc
tgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct
ccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgttt
ggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagt
aagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct
gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcg
gggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagc
aaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgt
ctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatga
cattaacctataaaaataggcgtatcacgaggccctttcgtc
R. E.s and DNA Ligase
Can be used to make recombinant DNA
EcoRI
EcoRI
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
1 Digestion
AATTC
G
2 Annealing of sticky ends
Ligase
G AATTC
CTTAA G
3 Ligation
4 Recombinant DNA
G AATTC
CTTAA G
Cloning Into pUC18
Ampr
pUC18
R. E.
Digestion
R. E.
Digestion
LacZ
Matching sticky
ends anneal
Host Cell
Transformation
of cells with the
recombinant
plasmid
Addition of ligase
joins nicks and
makes a single
recombinant
plasmind
So How Do You Know If
You Cloned Something?
IPTG - Induces
expression of lacZ
X-Gal - A lactose analog
which turns blue when
split by b-galactosidase
Ampicillin - Kills all
bacteria that lack the
plasmid
X-Gal
5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside
HOCH2
HOCH2
O
O
Galactose
HO
HO
Glucose
O
OH
HO
OH
OH
Lactose
O-b-D-galactopyranosyl-(1->4)-b-D-glucopyranose
X-Gal
5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside
b-Galactosidease
Lac Z
gene product
HOCH2
O
Galactose
HO
HO
O
(Colorless)
Cl
Br
OH
X-Gal
H2O
N
H
X-Gal
5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside
b-Galactosidease
HOCH2
O
Cl
Galactose
HO
HO
OH
HO
Br
OH
N
H
Blue
So How Do You Know If
You Cloned Something?
Blue colonies - Express b-galatosidase
IPTG - Induces
expression of lacZ
X-Gal - A lactose analog
which turns blue when
split by b-galactosidase
Ampicillin - Kills all
bacteria that lack the
plasmid
which metabolizes colorles X-gal to blue
and turn blue thus lacZ is not disrupted
Cloned fragments
and there is no foreign DNA cloned
disrupt lacZ thus make
no b-galactosidase and
colonies remain white
Libraries
1.
2.
3.
4.
5.
If all the DNA from an organism is digested with a
restriction enzyme and cloned into a plasmid, many
different recombinant plasmids will be made, each with
a different fragment of DNA cloned into it
Once inserted into host cells or viruses, this collection of
many different recombinant plasmids is called a
“library”
When the whole genome of an organism is used as the
starting point for cloning, it is called a “shotgun clone”
A library constructed using shotgun cloning may contain
hundreds of thousands of different recombinant plasmids
Screening is the process of sifting through the library to
find the clone of interest
A Library
The clone of interest
Strategy
Extract DNA
Fragment DNA
Insert into vector
DNA Library in bacteria
Screen library
and grow up bacteria with clone of interest
Library Screening
1.
2.
3.
4.
5.
Libraries tend to have a lot of clones only one of which
has the sequence of interest
Screening a library is the process of eliminating those
clones that do not contain the sequence of interest and
locating the clone that does
There two major techniques are used for screening:
Hybridization screening - In which DNA from a library
is bound to a membrane, then the membrane is exposed
to a probe that should base pair (hybridize) to the
sequence of interest
Expression vectors may be used so that if the gene for a
protein is cloned, the protein is made. To do this, you
must be able to detect the protein
cDNA Libraries
1.
2.
3.
4.
5.
Because of the large size of libraries and the tedium of
screening, anything that can be done to limit library
size is a good thing
Protein coding regions of most eukaryotic genomes
make up only a small percentage of the total DNA (3%
in humans)
Most cells only express a small subset of an organism’s
genes
By using reverse transcriptase, a cDNA copies of the
mRNA being produced in a group of cells can be made
Cloning cDNA to make a library produces a much
smaller library enriched with the part of an organism’s
genome that is of most interest
An Expression Vector
Myc tag
EK site
MCS
AatII
II
Cmr
I
XbaI
T1
pPROTet.E
II
t0
SacI
ColE1
• pPROTet.E is a
commercially
available plasmid sold
by Clontech
• It is specifically
designed to allow
efficient control of
expression
cDNA Library Construction
mRNA
5’
Reverse transcription
5’
mRNA Rev.
cDNA Trans.
hybrid 5’
5’
AAAAAAAAAAA3’
TTTTTTTTTTTT5’
AAAAAAAAAAA3’
TTTTTTTTTTTT5’
AAAAAAAAAAA3’
cDNA after RNase treatment
5’
RN
TTTTTTTTTTTT5’
ase
Double stranded cDNA after DNA polymerase
5’
5’
Insert into vector
DNA
Pol
TTTTTTTTTTTT5’
AAAAAAAAAAA3’