Aula BMol 4

Cloning vectors
E. coli
Yeast
Plants
Vectors for gene cloning
To be able to act as a vector for gene cloning, a DNA molecule
must:
-be able to replicate within the host cell so that numerous copies of
the recombinant DNA molecule can be produced
-be relatively small (<10 kb), as large molecules tend to break

during purification
-have high transformation efficiency
-have selectable markers
-ability to clone reasonably large (up to 8 kb) pieces of DNA
Two kinds of DNA molecules satisfy these criteria: plasmids and

bacteriophage chromosomes
Plasmids
- Plasmids are circular molecules of DNA that lead an independent
existence in the bacterial cell
- They carry one or more genes responsible for useful

characteristics displayed by the host bacterium
The ability to survive in normally
toxic concentrations of antibiotics is
often due to the presence in the
bacterium of a plasmid carrying
antibiotic resistance genes
In laboratory, antibiotic resistance is

often used as a selectable marker to
ensure that bacteria in a culture
contain a particular plasmid
Plasmids possess a DNA sequence that act as an origin of
replication, so that they multiply independently of the main
bacterial chromosome
Size and copy number
The copy number can be as low as one or as many as 100 or more.
Large plasmids have stringent control o replication; small plasmids

have it relaxed.
Useful cloning vectors need to be present in the cell in multiple

copies so that large quantities of the recombinant DNA molecule can
be obtained
Conjugation and compatibility
To be able to coexist in the

same cell, different plasmids
must be compatible. If two
plasmids are incompatible
then one or the other will be
rapidly lost from the cell
Plasmid classification:
-Fertility of F plasmids
-Resistance or R plasmids
-Col plasmids
-Degradative plasmids
-Virulence plasmids
Cloning vectors
The simplest cloning vectors are based on small bacterial
plasmids
Desirable properties:
-Easy purification
-High transformation efficiency
-Selectable markers
-Ability to clone reasonably large (up to about 8 kb)

pieces of DNA
Cloning vectors for E. coli
One of the first vectors to be developed was pBR322
-Small size (4363 bp)
-Two selectable markers

(antibiotic resistance genes,
ampicillin and tetracycline)
-Unique restriction sites for

cloning
-Reasonably high copy number

(15 copies/cell)
- Conjugative plasmid
The origins of pBBR322
Selecting cells that contain pBR322 plasmids
Identification of recombinants by insertional inactivation
How to distinguish
between cells that have
recombinant DNA
molecules from the ones
that contain self-ligated
vector?
Screening for
pBR322
recombinants by
insertional
inactivation of the
tetracycline
resistance gene
The pUC plasmids
-high copy number

(500-700 per cell)
-identification of
recombinant cells can
be achieved in one
step
-a multicloning site
clustered and
allowance to clone
fragments with two
different sticky ends
b-galactosidase splits
lactose into glucose and
galactose, but the
lactose analogue X-gal
(5-bromo-4-chloro-3-
indolyl-b-D-
galactopyranoside)
which is broken down by
b-galactyosidase
produces a coloured blue
compound
-cells producing b-
galactosidase turn blue
Bacteriophages
Virus that specifically infect bacteria
Simple structure, consisting merely of a DNA molecule and a

protective coat or capsid made up of proteins
l phage M13
The lysogenic infection cycle of
bacteriophage lambda (l)
The integrated form of the phage

DNA is called the prophage, and
the bacterium that carries it the
lysogen
The linear and circular forms of l phage
The l phage has two single-stranded cohesive ends called the
cos sites.
They allow the circularization of the linear molecule when
present in the cell and the cut, by an enzyme, of single phage
units from the catenane formed during phage replication
Long DNA fragments can be cloned using a
cosmid
Cosmids are hybrids between a phage DNA molecule and a

bacterial plasmid, and their design centres on the fact that the
enzymes that pack the l DNA molecule into the phage protein
coat need only the cos sites in order to function
The in vitro packaging reaction works with any molecule that

carries cos sites separated by 37-52 kb of DNA
A typical cloning with a cosmid
l and other high capacity vectors enable genomic
libraries to be constructed
A genomic library is a set of recombinant clones that contain

all of the DNA present in an individual organism
Genomic libraries can be retained for many years and copies

can be sent from one research group to another
How many clones are need for a genomic library?
N- number of clones required

ln (1-P) P- probability that any given gene will be
N= ln (1-a/b)
present
a- the average size of the DNA fragments
inserted into the vector
b- the total size of the genome
Insert capacities for common vector
systems
Cloning vectors for yeast
The yeast Saccharomyces

cerevisiae is very important in
Biotechnology: brewing,
breadmaking and a host
organism for the production of
many pharmaceuticals
The development of cloning

vectors was accelerated by the
discovery of the 2 mm plasmid Small plasmid
70-200 copies/cell
Plasmid origin of replication
Selectable markers for the 2 mm plasmid
Yeast episomal plasmids (YEps)
YEps are called shuttle vectors because they can replicate
both in E. coli and S. cerevisiae
-2 mm origin of replication
-Selectable LEU2 gene
-Entire pBR322 sequence
-copy number between 20-50
YEp13
Cloning vectors for higher plants
Vectors based on naturally occuring plasmids of Agrobacterium
Ti (tumor inducing)
The binary vector strategy
The Ti plasmid has a large size and the genetic manipulation

is very difficult
The T-DNA does not need to be physically attached to the rest

of the Ti plasmid
The only parts of the T-DNA that are involved in the infection
process are two 25 bp repeats found at the left and right
borders of the T-DNA
pBIN19
binary vector
(disarmed Ti)
Limitation of cloning with Agrobacterium plasmids:

-only for dicotyledonous plants (tomato, tobacco, potato, peas,
beans)

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Cloning vectors

-be relatively small (<10 kb), as large molecules tend to break

-have high transformation efficiency

-have selectable markers

-ability to clone reasonably large (up to 8 kb) pieces of DNA

Two kinds of DNA molecules satisfy these criteria: plasmids and

- They carry one or more genes responsible for useful

In laboratory, antibiotic resistance is

The copy number can be as low as one or as many as 100 or more.

Large plasmids have stringent control o replication; small plasmids

Useful cloning vectors need to be present in the cell in multiple

To be able to coexist in the

-High transformation efficiency

-Ability to clone reasonably large (up to about 8 kb)

-Small size (4363 bp)

-Two selectable markers

-Unique restriction sites for

-Reasonably high copy number

-high copy number

Simple structure, consisting merely of a DNA molecule and a

The integrated form of the phage

Cosmids are hybrids between a phage DNA molecule and a

The in vitro packaging reaction works with any molecule that

A genomic library is a set of recombinant clones that contain

Genomic libraries can be retained for many years and copies

How many clones are need for a genomic library?

N- number of clones required

The yeast Saccharomyces

The development of cloning

Vectors based on naturally occuring plasmids of Agrobacterium

The Ti plasmid has a large size and the genetic manipulation

The T-DNA does not need to be physically attached to the rest

Limitation of cloning with Agrobacterium plasmids:

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