Lecture 8:

Vectors based on the lambda

bacteriophage, cosmids and supervectors

Revision: Plasmid Vectors

Convenient for cloning of small DNA fragments
for restriction mapping and for studying
regulatory regions
Relatively small insert capacity
Handling and storage of these clones is timeconsuming and difficult (the repeated
subcultures of recombinants may result in
deletions in the inserts)

Revision: Plasmid Vectors

Plasmid vectors can be of three main types:
1. General purpose cloning vectors.

Cloning of foreign DNA fragments in general-purpose

cloning vectors selectively inactivates one of the
markers (insertional inactivation) or derepresses a
silent marker (positive selection) so as to differentiate
the recombinants from the native phenotype of the
vector

2. Expression vectors

DNA to be cloned and expressed is inserted

downstream of a strong promoter present in the
vector

3. Promoter probe and terminator probe

vectors

Useful for isolation of regulatory sequences such

as promoters and terminators and for studying
their recognition by a specific host

Cloning Vectors Based On

Lambda Bacteriophage

Lambda biology
.

The lambda virus particle contains a linear DNA

of ~48.5 kb with a single stranded 5 extension
of 12 bases at both ends which are
complementary to each other (called cohesive
ends or cos)
During infection, the right 5 extension (cosR),
followed by the entire genome, enters the host
cell
Both of the cos ends are ligated by E. coli ligase,
forming a covalently circular DNA

cohesive ends or
cos

cohesive ends or
cos

Vector based on bacteriophage lambda allow

efficient cloning of larger fragments, which is
important in constructing gene libraries

Development of Lambda:
2 Alternatives Modes
1. Lytic Cycle:

After infecting the host, the lambda genome may

start its replicationresults in the formation of
multiple copies of the genome

2. Lysogeny

The phage genome may enter a dormant stage

(prophage) by integrating itself onto a bacterial
genome by site-specific recombination; during this
stage it is propagated along with the host in the
subsequent progeny

Development of Lambda:
2 Alternatives Modes

 The lytic cycle:

is a productive cycle
that resulting in lysis of
the E. coli cell and
liberation of a number
of phage particles

Thy lysogeny cycle:

The lambda genome is
able to integrate into the
bacterial chromosome,
Where it can remain
quiescent for many
generations,
Being replicated along
with the host
chromosome every time
the cell divides

 The proportion of infected cells going down each route is

influenced by:
i. Environmental condition
ii. Genetic composition of the phage and the host

The broad objectives in constructing various phage vectors are:

i. the presence of cloning sites only in the dispensable fragments
ii. the capacity to accommodate foreign DNA fragments of various
sizes
iii. the presence of multiple cloning sites
iv. an indication of incorporation of DNA fragments by a change in
the plaque type
v. the ability to control transcription of a cloned fragment from
promoters on the vector
vi. the possibility of growing vectors and clones to high yield
vii.easy and ready recovery of cloned DNA
viii.introduction of features contributing to better biological
containment

Two types of vector have been developed:

1. Insertion Vectors:
i.

Cloning of foreign DNA exploits the insertional

inactivation concept

ii.

Possesses at least one unique restriction site into

which new DNA can be inserted

iii.

Example: gt10 / ZAPII

Insertion Vectors

2. Replacement Vectors:
i.

A large segment of the non-essential region (stuffer)

has been deleted, and the two arms ligated together

ii.

Two recognition sites for the RE used for cloning.

iii.

These sites flank a segment of DNA that is replaced

by the DNA to be cloned.

iv.

Example: WES.B / EMBL4

Replacement Vectors

Cloning with lambda vector

1. Ligation of each arm and the DNA to be cloned
2. Produce concatamers: left arm new DNA right arm.
3. The concatamers are then added to an in vitro packaging mix,
which contains all the proteins needed to make a lambda phage
particle
4. These proteins form phage particles spontaneously and place
inside the particles any DNA fragment that is between 37 to 52 kb
in length and is flanked by cos sites
5. The packaging mix cuts left arm new DNA right arm
combinations of 37 52 kb out of the concatamers and constructs
lambda phages around them
6. Infection of E. coli process transports the vector plus new DNA
into the bacteria

A concatamer is a long continuous

DNA molecule that contains multiple
copies of the same DNA sequences
linked in series

In vitro
packaging

7. After infection, the cells are spread onto an agar plate

8. Produce an even layer of bacteria across the entire
surface of the agar
9. Bacteria that were infected die within about 20 min
10. Death and lysis of the bacterium releases new phages
into the surrounding medium, where they infect new
cells and begin another round of phage replication and
lysis
11. The end result is a zone of clearing, called a plaque,
which is visible on the lawn of bacteria that grows on the
agar plate
12. Ligation of the two arms without insertion of new DNA
results in a molecule that is too short to be packaged

COSMIDS:
Vector for longer pieces of
DNA

Cosmids are plasmids containing lambda cos

ends

4 to 6 kb in size and are specifically designed for

cloning large DNA fragments (40-50 kb)

Cosmids have:
i. Drug resistance marker
ii. A plasmid origin of replication
iii. A fragment carrying the ligated cohesive ends
(cos) of phage lambda
iv. One or more unique restriction sites for cloning

Structure of a cosmid
One or more unique
restriction sites for
cloning
Drug resistance
marker

A fragment carrying
the ligated cohesive
ends (cos) of phage
lambda
A plasmid origin of
replication

Subject the ligation mixture to in vitro packaging

(insert should be between ~32 and 45 kb)

Concatamers of cosmid molecules, linked at their cos

sites, act as substrates for in vitro packaging

The cos site is the only sequence that a DNA

molecule needs in order to be recognized as a
lambda genome by the proteins that package DNA
into lambda phage particles

Particles containing cosmid DNA are as infective as

real lambda phages, but once inside the cell cannot
direct synthesis of new phage particles and instead
replicates as a plasmid

Supervectors:
YACs and BACs

Supervectors
YAC: yeast artificial chromosome
Able to carry 100 kb or more up to 2 Mb (~1000 x more
DNA insert than in a plasmid)
Propagated as circular plasmid in E.coli

BAC: bacterial artificial chromosome

Able to carry inserts of 300 kb
Based on F plasmid (The prototype conjugative plasmid
associated with conjugation in a strain of E. coli)
Important in genome sequencing project

Structure of a YAC vector

1.

2.

3.
4.

5.

BamHI removes the

stuffer fragment between
the two telomers
SnaBI cuts the vector into
two linear arms (each
carrying a selectable
marker)
Insert is ligated between
these arms
Transform into a yeast cell
(auxotrophic markersensures that the
recombinants contain
both arms)

Successful recombinant must contain the tel sequences at each end so

that the yeast transformant can use these sequences to build functional
telomers

Illustration of a chromosome

End of Lecture 8
Thank You