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Cloning Vectors

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 Plasmid Vectors
• Circular, double-stranded circular DNA
molecules present in bacteria.
• Range from 1 kb to over 200 kb.
• Replicate autonomously (origin of replication).
• Many carry antibiotic-resistance genes, which
can be used as selectable markers.
• Many useful cloning vectors were derived
from plasmid pBR322.

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Successful cloning
is identified by
insertional inactivation
of one of the two
antibiotic resistance
genes

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 Amplification of Recombinant DNA
• Antibiotic-sensitive recipient cells are
transformed with the recombinant DNA
molecule.
• Transformed cells are selected by growth under
conditions requiring the presence of a
selectable marker present on the recombinant
DNA molecule (usually an antibiotic).
• The recombinant DNA molecule is amplified by
the host cell.

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Cloning can be in one
of the two resistance
genes. Results for
cloning in either
tetracycline or
ampiciline resistance
gene
Colonies contain + tet + amp
No plasmid - -
No insert but the + +
plasmid is present
Insert in the tet - +
resistance gene
Insert in the amp + -
resistance gene

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• Multiple cloning site (MCS)
• Blue-White Color Test
• Phage f1 origin
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 The Blue-White Color Test
• The E. coli lacZ gene encodes
-galactosidase.
• -galactosidase converts the
colorless substrate Xgal into
a blue product.
• Cloning an insert inside lacZ
gene stop production of -
galactosidase.
• Cells with -galactosidase
activity produce blue
colonies when grown on
Xgal; cells lacking -
galactosidase activity
produce white colonies.
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X-gal produce blue
color in presence of
the galctosidase

IPTG is lactose
analog the induce
Lac operon
 Bacteriophage Vectors
• Most bacteriophage cloning vectors have
been constructed from the phage 
chromosome.
• The central one-third (about 15 kb) of the 
chromosome contains genes required for
lysogeny but not for lytic growth.
• This portion of the chromosome can be
excised and replaced with foreign DNA.
• The foreign DNA inserted must be 10-15 kb.

© John Wiley & Sons, Inc.

Phage Package?

Plaque?
 Cosmid Vectors
• Hybrids between plasmids and the phage 
chromosome.
• Replicate autonomously in E. coli.
• Can be packaged in vitro into phage  heads.
• Accept inserts of 35-45 kb.

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 Eukaryotic and Shuttle Vectors
• Because different organisms use different origins of
replication and regulatory signals, different cloning
vectors must be used in different species.

• Special cloning vectors can replicate in other

prokaryotes and in eukaryotes.

• Shuttle vectors can replicate in E. coli and in another

species.

© John Wiley & Sons, Inc.

Introduce DNA
into cells
without cell wall
either by
-Microinjection
or
-Electroporation
 Yeast Artificial Chromosomes (YACs)

• Genetically engineered yeast

minichromosomes.
• Accept foreign DNA inserts of 200-500 kb.
• Contain a yeast origin of replication, yeast
centromere, two yeast telomeres, a selectable
marker, and a polycloning site.

© John Wiley & Sons, Inc.

 BACs and PACs
BACs
• Bacterial artificial chromosomes (BACs) have been
constructed from bacterial fertility (F) factors.
PACs
• Bacteriophage P1 artificial chromosomes (PACs) have
been constructed from bacteriophage P1
chromosomes.

BACs and PACs accept 150-300 kb inserts and are less

complex than YACs.

© John Wiley & Sons, Inc.

 Expression vector
Vector including a promoter
from the target organism

1. Express heterologous or mutant genes to assess their function

2. Produce large amount of protein

3. Study gene regulation by addition of a reporter gene