Cloning Vectors in

Bacteria
Plasmids and Phages
Bacterial Strains and Plasmid Vectors
 Cloning Vectors
Recombinant DNA Molecule

Vectors:

Bacteria Yeast Plant Cell Animal Cell

A cloning vector is defined as a vehicle to carry a foreign DNA

sequence into a host cell where it can be replicated and or/expressed
Bacteria as hosts
eg.- Eschericia coli, Bacillus subtilis
1. They are easily grown
2. They are cheap to grow
3. They grow fast
4. They are easily manipulated in the laboratory
a) DNA can be inserted - transformation
b) DNA can be easily isolated
5. Non-pathogenic
6. Genetically stable
7. Bacteria contain natural plasmids and viruses which are
useful vectors for recombinant DNA
8. Equipped with appropriate enzymes to allow replication of
the vector
Bacterial Engineered Host Strains
• A few mutations are common to all or most expression strains to accommodate high protein
levels including:
• ompT: Strains harboring this mutation are deficient in outer membrane protease VII, which
reduces proteolysis of the expressed recombinant proteins.

• lon protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce
proteolysis of the expressed proteins.

hsdSB (rB- mB-): These strains have an inactivated native restriction/methylation system. This
means the strain can neither restrict nor methylate DNA.

• Dcm/Dam: Similarly, strains with this mutation are unable to methylate cytosine/adenine within a
particular sequence.
 Properties of a Cloning Vector
Important features for cloning vector;
1. Origin of replication (ori)- for independent
replication within the host
2. A selectable marker- for identifying the host cells
containing the vector, like- antibiotic resistance,
enzymes such as β-galactosidase, GFP, etc.
3. Multiple Cloning Site (MCS) or polylinker- for cutting
the vector and introducing an insert with the help
of restriction enzyme recognition sites. MCS
provides flexibility in the choice and use of
restriction enzymes.
4. Cloning vector should be small in size for easier
entry/transfer into a host cell
5. Expression vector- also has a promoter sequence
for driving expression of the transgene
A range of cloning vectors have been developed incorporating these features

Plasmid
Bacteriophage

Virus

Cosmids

Bacterial Artificial Chromosomes

(BAC)
Yeast Artificial Chromosomes
(YAC)
Vectors for cloning in Bacteria

Bacterial Plasmids Bacteriophages

 Bacterial Plasmids
₋ Extrachromosomal
₋ Self-replicating
₋ Usually circular, double-stranded DNA
molecules
₋ Found naturally in many bacteria and some
yeasts
₋ Size ranges from 5-400kb
₋ Confer useful properties to the host, like
resistance to antibiotics, toxin production, etc.–
Selective advantage
₋ Present as 1 or 2 copies or in multiple copies
(500-700) inside the host
 Plasmid Cloning Vectors
Naturally occurring plasmids have been modified to serve
as vectors in the lab and are widely used, versatile and
easily manipulated vectors
Important features for cloning vector;
1. Origin of replication (ori)- for independent
replication within the host
2. A selectable marker- for identifying the host cells
containing the vector, like- antibiotic resistance,
enzymes such as β-galactosidase, GFP, etc.
3. Multiple Cloning Site (MCS) or polylinker- for cutting
the vector and introducing an insert with the help
of restriction enzyme recognition sites. MCS
provides flexibility in the choice and use of
restriction enzymes.
4. Cloning vector should be small in size for easier
entry/transfer into a host cell
 Origin of Replication
An origin of replication (ori) is a sequence of DNA (generally AT-rich) at which replication is initiated on
a chromosome, plasmid or virus. For small DNAs, including bacterial plasmids and small viruses, a
single origin is sufficient.
 Origin of Replication and Copy number
Common Copy ori Control Incombatibility
Vectors Number Groups
pBR322 ~15-20 pMB1 Relaxed A
pUC ~500-700 pMB1 Relaxed A
(derivative)
pColE1 ~15-20 ColE1 Relaxed A
pSC101 ~5 pSC101 Stringent C

pACYC ~10 p15A Relaxed B

pBluescrip ~300-500 ColE1 Relaxed A
t (derivative) and
F1

pGEM ~300-500 pUC and F1 Relaxed A

High copy number = 10-500 copies / cell  generally Non conjugative, relaxed
Low copy number = 1-5 copies / cell  generally Conjugative, HMW, stringent
Note: the pMB1 ori maintains about 20 copies per cell, while pUC – which differs by only two
mutations – will produce as many as 700 copies per cell.
Plasmid Incompatibility and Host Range
Plasmid Incompatibility Plasmid Host Range
Plasmid incompatibility refers to the inability Narrow Host Range- Most of the naturally
of two plasmids to coexist stably over a occuring plasmids have a restricted host
number of generations in the same bacterial range eg. ColE1 and its derivates can grow
cell line. only in E. coli and related bacteria like
Generally, closely related plasmids tend to Salmonella
be incompatible, while distantly Broad Host Range – plasmids can be
related plasmids tend to be compatible. transferred and replicated in a broad range of
bacteria eg. RP4 and RSF101

Plasmids Compatible Plasmids incompatible,

Normal replication, Low replication frequency
plasmids are maintained Plasmids may be lost
Shuttle Vectors
Shuttle vectors are the engineered plasmids containing two origin of replication so
that they can multiply in two different host species.
Shuttle Vectors have also been developed that can exist both in the eukaryotic cell and
E. coli. They contain two types of ori and selectable marker genes, for each eukaryotic
cells (e.g. yeast) and E. coli. Example; YEp, Ti plasmid in case of plants.
Selectable or Screenable Markers

The gene expressing Green Fluorescent

Protein (GFP) causes host cells
containing the vector to fluoresce when
Blue color formation by enzyme beta- viewed under UV light.
Selecting transformed cells resistant galactosidase (β-gal) activity on substrate X-
to antibiotics, like ampicillin, by gal, and its application in blue/white
growing cells in medium containing screening.
the antibiotic.
 Markers (Antibiotic resistance)
Ampicillin (beta-lactam class) inhibits synthesis of bacterial cell wall
amp resistance depends upon production of an enzyme that catalyzes beta-
lactam ring degradation in the periplasmic space.

Tetracycline binds to 30S subunit of the ribosome to prevent ribosome

translocation
tet resistance produces a protein that prevents tetracycline from entering the
cell.

Chloramphenicol binds to the 50S subunit of the ribosome to prevent protein

synthesis
cat (chloramphenicol resistance) produces an enzyme system component that
converts chloramphenicol to a form that cannot bind the ribosome.

Kanamycin (and the closely related neomycin) are aminoglycosides that bind
sub-components of the ribosome and prevent protein synthesis.
kan (and neo) resistance depend upon the synthesis of an aminoglycoside
phosphotransferase located in the periplasmic space that inhibits their
transport into the cell.
Selectable or Screenable Markers
Selecting transformed cells resistant to
antibiotics, like ampicillin, by growing cells
in medium containing the antibiotic.

Blue color formation by enzyme beta-

galactosidase (β-gal) activity on substrate X-
gal, and its application in blue/white
screening.

The gene expressing Green Fluorescent

Protein (GFP) causes host cells containing the
vector to fluoresce when viewed under UV
light.
 pBR322- One of the earliest plasmid cloning vector
pBR322 comprises DNA derived from three different naturally occurring plasmids.

The ampR gene originally resided on The tetR gene is derived from R6-5,
the plasmid R1, a typical antibiotic a second antibiotic-resistant
resistance plasmid that occurs in plasmid.
natural populations of E. coli

~15 copies/cell (may be increased to ~1000 by

use of chloramphenicol)

The ori is originally from pMB1, which is closely

related to the colicin-producing plasmid ColE1.

The name “pBR322” conforms with the standard rules for vector nomenclature:
“p” - plasmid.
“BR”- BR stands for Bolivar and Rodriguez, the two researchers who developed pBR322.
“322” distinguishes this plasmid from others developed in the same laboratory
(there are also plasmids called pBR325, pBR327, pBR328,
Screening bacteria by replica plating
Eg. The cloning is performed in Bam HI Site
 pUC vector series (pUC18/19)- Advanced Vectors
Inclusionof Polylinker (also called multiple cloning site) of ~ 60
bases which has sites for multiple restriction endonucleases

Have a region of the lacZ gene that codes for β-

galactosidase. This region also contains a polylinker. Shorter than pBR322 (due to removal
Thus, insertion of a foreign DNA into any of the
of most of non-essential DNA) and thus
restriction sites results in an altered non-functional
can clone larger fragments
enzyme. Thus, facilitates Blue-White Screening

The name “pUC” conforms with the standard

rules for vector nomenclature:
“p” - plasmid.
“UC”- stands for University of California where
early work on the vector was conducted
The polylinker
Unique sites (usually)


advantage
Insert excision facilitated


Restriction endonuclease mapping and Subcloning



made easier
 Directional cloning
 Blue White Screening-Alpha complementation:
LacZ Beta galactosidase
(Homotetramer)
1021aa

• Bacteria carry mutant allele (LacZΔM15)

lacking N-terminal domain  inactive
protein

• Alpha peptide carried by plasmid

• Exploits X-Gal (5-bromo-4-cloro-3-indolil-

Betagalattoside), a chromogenic substrate
analog to galactose

• MCS inserted into LacZ alpha peptide 

• With insert = white colonies

• Without insert = blue colonies
Another Major Advance: Blue-White Screening
The blue colonies contain “self” religated plasmids that do not
have DNA inserts interrupting the lac Z gene.

White colonies consist of bacteria that carry plasmids that have

DNA insert fragments that interrupt the lac Z gene.

Beware False Positives:

Blue-white screening only indicates the presence of AN insert,
not necessarily YOUR insert.
pGEM Vectors- Expression Vectors
Introduction of a promoter for in vitro transcription of cloned DNA
pET vectors: protein expression
 Commonly Used Bacterial Strains
Strain Resistance Key Features Use
Basic IPTG-inducible strain containing General protein
BL21 (DE3)
T7 RNAP (DE3) expression

pLysS expresses T7 lysozyme to reduce

Chloramphenicol basal expression levels; expression Expression of toxic
BL21 (DE3) pLysS*
(pLysS) vector cannot have p15A origin of proteins
replication

pLysE has higher T7 lysozyme

Chloramphenicol expression than pLysS; expression Expression of toxic
BL21 (DE3) pLysE*
(pLysE) vector cannot have p15A origin of proteins
replication

General expression; not

Lacks functional RNaseE which results
BL21 star (DE3) recommended for toxic
in longer transcript half-life
proteins

RecA-deficient; best for plasmids with Expression of unstable

BLR (DE3) Tetracycline
repetitive sequences. proteins

E. coli BL21(DE3), a derivative of BL21, is probably the most widely used in high-level expression of
recombinant proteins, and it harbors a prophage DE3 derived from a bacteriophage λ, which carries
the T7 RNA polymerase gene under the control of the lacUV5 promoter. lacUV5 is very similar to the
classical lac promoter, containing just 2 base pair mutations in the -10 hexamer region, compared to
the lac promoter. LacUV5 is among the most commonly used promoters in molecular biology because it requires
no additional activators and it drives high levels of gene expression.
 Bacterial Strains
Strain Resistance Key Features Use
Contains highly active
thioredoxin reductase
and glutathione
Streptomycin and Expression of insoluble
Origami2 (DE3)** reductase to faciliate
Tetracycline proteins
proper folding; may
increase multimer
formation
Good for “universal”
translation; contains 7
additional tRNAs for rare
Chloramphenicol codons not normally Expression of eukaryotic
Rosetta2 (DE3)*
(pRARE) used in E. proteins
coli. Expression vector
cannot have p15A origin
of replication
Rhamnose-tunable T7
RNAP expression
Expression of toxic,
Chloramphenicol alleviates inclusion body
Lemo21 (DE3)* insoluble, or membrane
(pLemo) formation. Expression
proteins
vector cannot have p15A
origin of replication
Factors required for high-level expression of genes

1) transcriptional promoter & terminator

2) Shine Dalgarno Sequence (ribosome binding site)
3) Efficiency of translation
4) Stability of the protein
5) Final cellular location (secreted?)
6) Number of copies of cloned gene
7) Host organism
 E. coli Promoter
Sites

Deviation from consensus -10 , -35 sequence leads to

weaker gene expression
Promoters of Importance in
Biotech
tac (trc) promoter
• Hybrid of lac and trp promoters
-35 region from trp
-10 region from lac
• separated by 16bp = tac promoter
• separated by 17bp = trc promoter

• 3x stronger than trp

• 5-10x stronger than lac
 Shine Dalgarno Sequence
An efficient expression vector requires not only a strong, regulatable promoter, but also an E.
coli ribosome binding sequence and a terminator.

• In Prokaryotes
– translation signal is RBS
– ribosome binding site
– Shine-Dalgarno sequence (AGGAGGU)
– 6-8 nt in length
– located short distance (~10nt) upstream of
AUG translation start codon
Limitations with plasmid vectors
 Limit of DNA size cloning is ~10 kb

 Transformation efficiency

 Limitations regarding the expression of a eukaryotic

protein expression