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 Cosmids, Phagemids, Bacterial
Artificial Chromosomes (BACs), Yeast Artificial
Chromosomes (YACs) and Human Artificial
Chromosomes (HACs).
 Plasmid vectors
Plasmid vectors are double-stranded, circular, self-
replicating, extra-chromosomal DNA molecules.

 Advantages:
– Small, easy to handle
– Straightforward selection strategies
– Useful for cloning small DNA fragments
(< 10kbp)
 Disadvantages:
– Less useful for cloning large DNA fragments
(> 10kbp)
 Plasmid vectors

 Plasmids are circular DNA

molecules present in the
cytoplasm of the bacteria
 Capable of autonomous
replication
 Can transfer genes from one
cell to other
 Act as vectors in genetic
engineering.
 Can also present in Yeasts
 Plasmid vectors
 may encode genetic information for properties

1 Resitance to Antibiotics
2 Bacteriocins production
3 Enterotoxin production
4 Enhanced pathogen city
5 Reduced Sensitivity to mutagens
6 Degrade complex organic molecules

T.V.Rao MD
Cloning into a Plasmid
 Features of many modern Plasmids

•Small size
•Origin of replication
•Multiple cloning site (MCS)
•Selectable marker genes
•Some are expression vectors and have sequences
that allow RNA polymerase to transcribe genes
•DNA sequencing primers
 Plasmid
Plasmid vector
vector for
for cloning
cloning

1. Contains an origin of replication, allowing for

replication independent of host’s genome.
2. Contains Selective markers: Selection of cells
containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
A more detailed look at plasmids

Promotor
Site
Origin of
Replication

Antibiotic Multiple
Resistance Cloning
Gene Site
 Some antibiotics commonly used as selective
agents
Antibiotic Description
Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by -
lactamase, which cleaves the -lactam ring of amp
Hygromycin B An aminoglycoside that kills bacteria, fungi and
(HygB) higher eukaryotic cells by inhibiting protein synthesis;
inactivated by hygromycin B phosphotransferase (hph)
Kanamycin (Kan) Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a phosphotransferase
Neomycin (Neo) Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a phosphotransferase
Streptomycin (Str) Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by phosphotransferases (strA &
strB)
Tetracycline (Tet) Binds to 30S ribosomal subunit and inhibits protein
synthesis; tetr gene encodes a protein which prevents
transport of tet into the cell
 pBR322

ori

The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in length and
contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid pMB1); (2) rop gene
coding for the Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable
complex and serves to decrease copy number (source – plasmid pMB1); (3) bla gene, coding for beta-lactamase
that confers resistance to ampicillin (source – transposon Tn3); (4) tet gene, encoding tetracycline resistance
protein (source – plasmid pSC101).
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.
pUC18/19
pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp
in length. They are identical except that they contain multiple cloning sites (MCS)
arranged in opposite orientations. pUC18/19 plasmids contain: (1) the pMB1
replicon rep responsible for the replication of plasmid (source – plasmid pBR322).
The high copy number of pUC plasmids is a result of the lack of the rop gene and
a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase
that confers resistance to ampicillin (source – plasmid pBR322); (3) region of
E.coli operon lac containing CAP protein binding site, promoter Plac, lac
repressor binding site and 5’-terminal part of the lacZ gene encoding the N-
terminal fragment of beta-galactosidase (source – M13mp18/19). This fragment,
whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa)
complementation with a defective form of beta-galactosidase encoded by host
(mutation lacZDM15). In the presence of IPTG, bacteria synthesize both
fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of
DNA into the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced
by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes
alfa-complementation. Bacteria carrying recombinant plasmids therefore give rise
to white colonies.
pGEM-3Z
The Major Limitation of Cloning in Plasmids
 Upper limit for clone DNA size is 12 kb
 Requires the preparation of “competent” host cells

 Inefficient for generating genomic libraries as overlapping

regions needed to place in proper sequence
 Preference for smaller clones to be transformed
 If it is an expression vector there are often limitations
regarding eukaryotic protein expression
 Phage Cloning Vectors
 Fragments up to 23 kb can be may be accommodated by a phage vector
 Lambda is most common phage
 60% of the genome is needed for lytic pathway.
 Segments of the Lambda DNA is removed and a stuffer fragment is put in.
 The stuffer fragment keeps the vector at a correct size and carries marker
genes that are removed when foreign DNA is inserted into the vector.
 Example: Charon 4A Lambda
 When Charon 4A Lambda is intact, beta-galactosidase reacts with X-gal and
the colonies turn blue.
 When the DNA segment replaces the stuffer region, the lac5 gene is missing,
which codes for beta-galactosidase, no beta-galactosidase is formed, and the
colonies are white.
 Bacteriophage vectors

 Advantages:
– Useful for cloning large DNA fragments
(10 - 23 kbp)
– Inherent size selection for large inserts

 Disadvantages:
– Less easy to handle
 vectors

 Left arm:
– head & tail proteins
 Right arm:
– DNA synthesis
– regulation
– host lysis
 Deleted central region:
– integration &
excision
– regulation
 Cosmid Cloning Vectors
 Fragments from 30 to 46 kb can be accommodated by a
cosmid vector.
 Cosmids combine essential elements of a plasmid and
Lambda systems.
 Cosmids are extracted from bacteria and mixed with
restriction endonucleases.
 Cleaved cosmids are mixed with foreign DNA that has
been cleaved with the same endonuclease.
 Recombinant cosmids are packaged into lambda
caspids
 Recombinant cosmid is injected into the bacterial cell Shown above is a 50,000 base-pair long DNA
where the rcosmid arranges into a circle and replicates molecule bound with six EcoRI molecules, and
imaged using the atomic force microscope. This
as a plasmid. It can be maintained and recovered just as image clearly indicates the six EcoRI "sites" and
allows an accurate restriction enzyme map of
plasmids. the cosmid to be generated.
http://homer.ornl.gov/cbps/afmimaging.htm
 Cosmids
 Hybrid vectors: plasmids that contain
bacteriophage lambda cos sites
 DNA (~ 33-48 kb) cloned into ori
restriction site, the cosmid packaged 21.5 kb
into viral particles and these phages
TetR
used to infect E.coli
 Cosmid can replicate in bacterial cell,
so infected cells grow into normal cos
colonies
 Insert DNA limited by the amount of
DNA that can fit into phage capsule EcoRI
 Somewhat unstable, difficult to
maintain Cos site is the only
requirement for
packaging into
phage particle
 Cosmid
Combine the properties of plasmid vectors with
the useful properties of the l cos site
 Advantages:
– Useful for cloning very large DNA fragments
(32 - 47 kbp)
– Inherent size selection for large inserts
– Handle like plasmids
 Disadvantages:
– Not easy to handle very large plasmids
– (~ 50 kbp)
 Phagemid

ZAP
 Other Vectors
 BACs (Bacterial artificial chromosomes)
– Large low copy number plasmids (have ori and selectable marker)
– Can be electroporated into E. coli
– Useful for sequencing genomes, because insert size 100 - 300kb
 YAC (Yeast Artificial Chromosome)
– Can be grown in E.coli and Yeast
– Miniature chromosome (contains ori, selectable markers, two
telomeres, and a centromere
– Can accept 200 kb -1000 kb; useful for sequencing
 Ti plasmids; to introduce genes into plants
 Expression vectors
 BACs and YACs
BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
 Advantages:
– Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
– This is very important for genome sequencing
projects

 Disadvantages:
– Not easy to handle extremely large DNA
molecules
BAC vector

 oriS and oriE

mediate replication
 parA and parB
maintain single copy
number
 ChloramphenicolR
marker
 YAC vector
large
inserts

ARS URA3 HIS3

telomere centromere markers telomere
replication
origin

 Capable of carrying inserts of 200 - 2000 kbp in yeast

 Bacterial Artificial Chromosomes(BACs) and
Yeast Artificial Chromosomes(YACs)

 BACs can hold up to 300 kbs.

 YACs can hold up to 500 kbs.
 YACs are designed to replicate as plasmids in
 The F factor of E.coli is capable of bacteria when no foreign DNA is present.
handling large segments of DNA. Once a fragment is inserted, YACs are
 Recombinant BACs are introduced into transferred to cells, they then replicate as
E.coli by electroportation ( a brief high- eukaryotic chromosomes.
voltage current). Once in the cell, the  YACs contain: a yeast centromere, two yeast
rBAC replicates like an F factor. telomeres, a bacterial origin of replication, and
bacterial selectable markers.
 Example: pBAC108L  YAC plasmidYeast chromosome
 Has a set of regulatory genes, OriS, and  DNA is inserted to a unique restriction site,
repE which control F-factor replication, and cleaves the plasmid with another
and parA and parB which limit the restriction endonuclease that removes a
number of copies to one or two. fragment of DNA and causes the YAC to
 A chloramphenicol resistance gene, and a become linear. Once in the cell, the rYAC
replicates as a chromosome, also replicating
cloning segment. the foreign DNA.
Expression vector
Expression vector pSE420

• Polylinker: insert desired DNA

• Amp resistance

• trc promoter
• lacO (operator)
• Shine-Dalgarno (S/D) site
(ribosome binding)
• T1, T2 transcription terminators
• lacI (lac repressor)

cloned gene expressed;

growth inducer added product produced
What determines the choice vector?

 insert size
 vector size

 restriction sites

 copy number

 cloning efficiency

 ability to screen for inserts

 what down-stream experiments do you plan?

 Cloning vectors and their insert capacities
Vector system Host cell Insert capacity (kb)

Plasmid E. coli 0.1-10

Bacteriophage  E. coli 10-20

Cosmid E. coli 35-45

Bacteriophage P1 E. coli 80-100

BAC (Bacterial E. coli 50-300

Artificial Chromosome)
P1 bacteriophage- E. coli 100-300
derived AC
YAC Yeast 100-2,000
Human AC Cultured human cells >2,000
Some Commonly Used Molecular Cloning Techniques
Method Description
Cloning by PCR Generate restriction sites by PCR or Use TA Cloning

Restriction Enzyme Cloning Assemble plasmids by restriction digest and ligation

Gateway Recombination Fragment of DNA that is to clone into a plasmid must

Cloning already be surrounded by specific recombination sites
TOPO Cloning Topoisomerase based cloning
Gibson Assembly Combine overlapping DNA fragments in a single reaction
In-Fusion Cloning Allow ligation-independent cloning of PCR products into
any vector, at any site of linearization.
Type IIS Assembly (Golden Take advantage of the unique properties of type IIS
Gate & Modular Cloning) restriction endonucleases
Ligation Independent Scarless cloning that relies on 3'-5' exonuclease activity
Cloning (LIC) of T4 DNA polymerase
Yeast-mediated Cloning It takes advantage of the powerful recombination
and Oligonucleotide abilities of yeast. Another advantage is the ability to
Stitching perform oligonucleotide stitching, in which pieces of DNA
that share no end homology can still be fused together in
a seamless manner.
Invitrogen’s Gateway® technology facilitates cloning of genes, into and out of,
multiple vectors via site-specific recombination. Once a gene is cloned into an
Entry clone you can then move the DNA fragment into one or more destination
vectors simultaneously.