Lecture4 Vectors

How do scientists move DNA (genes)?
1. Isolate source of DNA
2. Join DNA to a vehicle that can transport it
3. Transfer DNA (from chromosomes) into target cell

Join DNA to a transport vehicle
Vector
– means by which DNA from one organism gets

to target/host cell
Types of Vectors
1. Mechanical
 Micropipette inserted into cell
 Gene gun shoots tiny metal bullet coated w/ DNA
2. Biological
 Virus
 Plasmid
VECTORS - Vehicles for
Cloning
- Essential features of cloning vectors
Cloning Vectors
 A vector is used to amplify a single molecule of
DNA into many copies. A DNA fragment must be
inserted into a cloning vector. A cloning vector is a
DNA molecule that has an origin of replication and
is capable of replicating in a bacterial cell.
 Most vectors are genetically engineered plasmids or

phages. There are also cosmid vectors, bacterial
artificial chromosomes, and yeast artificial
chromosomes.
Vector requirements
 Selectable marker
 Resistance to ampicillin, kanamycin, hygromycin, herbicide, etc.
 Allows only recombinant clones to survive
 Multiple cloning site (mcs)
 many (unique) restriction sites
 For vectors that amplify in E. coli cells:
 Origin of replication (Ori): required for plasmid to replicate in
bacterium
 Control of copy number (F factor plasmid): reduces copy number
of plasmids in a given cell, lessens problems of rearrangements.
 For vectors that amplify in yeast cells:
 Autonomous replication sequence (ARS): similar to Ori
 CEN: centromere
 TEL: telomere
Vectors
DNA molecule originating from a virus, a plasmid, or a cell of a higher
organism into which another DNA fragment of appropriate size can be
integrated without loss of the vector’s capacity for self-replication
Vectors introduce foreign DNA into host cells, where the DNA can be
reproduced in large quantities
Vectors Host - Features Insert Size

Plasmid E.coli- Introduced by transformation 1-5 kb
(electroporation or heat shock)
Phage E.coli- Virus that infects bacteria 10-15 kb
Introduced by transfection
Cosmid E.coli- Plasmid with “cos” sites for 30-45 kb
packaging into lambda phage particles.
Introduced by infection into E. coli
Cloning Vectors

Plasmid Insertion Replacement Cosmid
Insert size 0-10 kb 0-10 16-23 32-47
Insert 50 2-5 33-45 90
(% total DNA)
Cloning + +++ +++ +
efficiency
Ease of use +++ + + +
Isolation of +++ + + +
DNA
Plasmid Cloning Vectors
 Plasmids are circular, double-stranded DNA
molecules that exist in bacteria and in the nuclei of
some eukaryotic cells.
 They can replicate independently of the host cell.
The size of plasmids ranges from a few kb to near
100 kb
 Can hold up to 10 kb fragments
 Plasmids have an origin of replication, antibiotic
resistance genes as markers, and several unique
restriction sites.
 After culture growth, the clone fragment can be
recovered easily. The cells are lysed and the DNA
is isolated and purified.
 A DNA fragment can be kept indefinitely if mixed
with glycerol in a –70 degrees C freezer.
pBR322
A purpose-
built cloning
vehicle
construction
of in vitro
superior
cloning
vectors
86-1268  Tc
4084-3296 Ap
Ori  2535
Plasmids are Extrachromosomal
Self-Replicating DNA molecules
Plasmids are easy to purify
Features of plasmids:
1) Replication origin (ORI)—allows the plasmid to be replicated by the host
2) Selectable marker(s)—such as the ampicillin resistance gene permits for selection of the plasmid
3) Cloning site(s)—allows for the insertion of exogeneous DNA

Overview of Cloning a DNA Fragment into a Plasmid Vector
insert
Bacterial
Transformation
Plasmid Polylinkers and Marker Genes for Blue-
White screening
 A vector usually contains a sequence
(polylinker) which can recognize several
restriction enzymes so that the vector can be
used for cloning a variety of DNA samples.
 Colonies with recombinant plasmids are white,
and colonies with nonrecombinant plasmids are
blue.
 Example: pUC19
 Resistant to ampicillin, has (ampr gene)
 Contains portion of the lac operon which codes
for beta-galactosidase.
 X-gal is a substrate of beta-galactosidase and
turns blue in the presence of functional beta-
galactosidase is added to the medium.
 Insertion of foreign DNA into the polylinker
disrupts the lac operon, beta-galactosidase
becomes non-functional and the colonies fail to
turn blue, but appear white.
Properties of plasmid cloning vector
1. Small
2. Stable in the chosen
host – usually E. coli
3. High copy number
4. Easy to purify
5. Can accommodate
foreign DNA
6. Single “cloning” sites
7. Selectable marker –
antibiotic resistance
8. Easily introduced into
host (transformation or
transduction
-complementation – relies on modular structure
of -galactosidase
- basic idea is often used with cloning vectors – called insertional
inactivation
complementation
LacZ+ - blue colony M15 mutant

lac Z (11- 41 aa del)
LacZ- - white colony 3- 92 aa added in vitro
WT-1021 aa (3.1 kb gene)
-if you interrupt the lacZ
gene, the colony is white
How does -
complementation
work?
It all comes down to -

galactosidase
Certain strains supply the

-Gal fragment
When  is supplied in
trans, this allows -Gal to
function
Insertional Inactivation
LacZ gene
LacZ (-galactosidase)
Lactose Glucose + Galactose
X-gal X-gal metabolites
(clear) (blue)
5-bromo-4-chloro-3-
indolyl--D-galactoside
Non-recombinants (religated vector) produce blue colonies (LacZ +)
Recombinants (with an insert) produce white colonies (LacZ -)

Limitations of Plasmids
1) Can’t ligate large inserts into them (<10 Kb inserts)

2) Low efficiency of transformation
3) Large colony size
Can screen fewer recombinants per plate (~5,000 colonies /plate)

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How do scientists move DNA (genes)?

1. Isolate source of DNA

2. Join DNA to a vehicle that can transport it

3. Transfer DNA (from chromosomes) into target cell

– means by which DNA from one organism gets

 Most vectors are genetically engineered plasmids or

Vectors Host - Features Insert Size

Plasmids are easy to purify

3) Cloning site(s)—allows for the insertion of exogeneous DNA

LacZ+ - blue colony M15 mutant

It all comes down to -

Certain strains supply the

Non-recombinants (religated vector) produce blue colonies (LacZ +)

Recombinants (with an insert) produce white colonies (LacZ -)

1) Can’t ligate large inserts into them (<10 Kb inserts)

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