Lecture 3 Vectors II.pdf molecular biology

VECTORS

1. Bacteriophages as cloning vectors
By
Dr. Heba Hamdy Abouseada

2. 2. Bacteriophages or phages
The two main types of phage structure:
(a)head-and tail (e.g. λ).
(b)filamentous (e.g. M13).
λ Phage DNA M13 phage DNA
▪These are viruses that specifically infect bacteria
▪They consist mainly of a DNA (or occasionally an RNA) molecule carrying a
number of genes, surrounded by a protective coat or capsid made up of
protein molecules

3. 2. Phages: Infection cycle
1- The phage attaches to the bacterium and injects its DNA
2- The phage DNA molecule is replicated
3- Capsid components are synthesized, new phage
particles are assembled and released
The general pattern of infection of a
bacterial cell by a bacteriophage.

4. The general pattern of infection, which is the same for all
types of phage, is a three-step process:
1.The phage particle attaches to the outside of the bacterium
and injects its DNA chromosome into the cell.
2.The phage DNA molecule is replicated, usually by specific
phage enzymes coded by genes in the phage chromosome.
3.Other phage genes direct synthesis of the protein
components of the capsid, and new phage particles are
assembled and released from the bacterium.
With some phage types the entire infection cycle is
completed very quickly, possibly in less than 20 minutes.

5. Lytic infection cycle
•This type of rapid infection is called a lytic cycle, as
release of the new phage particles is associated with lysis
of the bacterial cell.
•The characteristic feature of a lytic infection cycle is that
phage DNA replication is immediately followed by
synthesis of capsid proteins, and the phage DNA molecule
is never maintained in a stable condition in the host cell.

6. 2. Phages: Infection cycle
1- The phage attaches to the bacterium and injects its DNA
2- The phage DNA molecule is replicated
3- Capsid components are synthesized, new phage
particles are assembled and released
The general pattern of infection of a
bacterial cell by a bacteriophage.
Called a Lytic infection cycle

7. Lysogenic infection cycle
•In contrast to a lytic cycle, lysogenic infection is characterized
by retention of the phage DNA molecule in the host
bacterium, possibly for many thousands of cell divisions.
•The integrated form of the phage DNA (called the prophage)
is quiescent, and a bacterium (referred to as a lysogen) that
carries a prophage is usually physiologically indistinguishable
from an uninfected cell.
•However, the prophage is eventually released from the host
genome and the phage reverts to the lytic mode and lyses the
cell.

8. •A limited number of lysogenic phages follow a rather different
infection cycle.
•When M13 or a related phage infects E. coli, new phage particles
are continuously assembled and released from the cell.
•The M13 DNA is not integrated into the bacterial genome and
does not become quiescent.
•With these phages, cell lysis never occurs, and the infected
bacterium can continue to grow and divide, at a slower rate than
uninfected cells.
Lysogenic infection cycle (M13)

9. 2. Phages: Infection cycle (Lysogenic)

10. 2. Phages: Infection cycle
Called a Lysogenic infection cycle
1- Bacteriophage λ 2- Bacteriophage M13

11. 2. Bacteriophages: λ Phage
The λ genetic map, showing the positions of the important genes and
the functions of the gene clusters.
(Gene organization in λ DNA molecule )

12. 2. Bacteriophages: λ Phage
Disadvantage 1
▪The λ DNA molecule can be increased in size by only about
5%, representing the addition of only 3kb of new DNA. If the
total size of the molecule is more than 52 kb, then it cannot
be packaged into the λ head structure and infective phage
particles are not formed.
This severely limits the size of a DNA fragment that can be
inserted into an unmodified λ vector.

13. 2. Bacteriophages: λ Phage
How to overcome?
•Large segment in the central region of the λ DNA molecule
can be removed without affecting the ability of the phage to
infect E. coli cells.
•Removal of all or part of this non-essential region, decreases
the size of the resulting λ molecule by up to 15 kb. This
means that as much as 18 kb of new DNA can now be added
before the cut-off point for packaging is reached.

14. 2. Bacteriophages: λ Phage
Disadvantage 2
▪The λ genome is so large that it has more than one
recognition sequence for virtually every restriction
endonuclease. Restriction cannot be used to cleave the
normal λ molecule in a way that will allow insertion of DNA.
▪Because, the molecule would be cut into several small
fragments would be very unlikely to re-form a viable X genome
on religation.

15. 2. Bacteriophages: λ Phage
Cosmids
•A cosmid is basically a plasmid that carries a fragment of λ DNA
including the cos site only (the only requirement for DNA to be
packaged into a phage particle.
•It also needs a selectable marker, such as the ampicillin
resistance gene, and a plasmid origin of replication.
•They can be used asgene-cloning vectors in conjunction with
the in vitro packaging system.

16. 2. Bacteriophages: M13- a filamentous phage
▪ Much smaller than λ DNA .
▪ Only 6407 nucleotides (6.4kb).
▪ Circular.
▪ Injected into E. coli cells as single-stranded DNA
molecule.

17. 2. Bacteriophages: M13- a filamentous phage
The M13 infection cycle, showing the different types of DNA replication that occur.
(a) Injection of single stranded DNA into
the host cell, followed by synthesis of the
second strand.
Double-stranded
DNA-replicative form (RF)

18. 2. Bacteriophages: M13- a filamentous phage
The M13 infection cycle, showing the different types of DNA replication that occur.
(b) Replication of the RF to produce new
double-stranded molecules

19. 2. Bacteriophages: M13- a filamentous phage
The M13 infection cycle, showing the different types of DNA replication that occur.
(c) Mature M13 phage are continuously produced.
RF replicates by rolling circle mechanism to
produce linear single-stranded DNA

20. 2. Bacteriophages: M13- a filamentous phage
•Although Ml3 vectors are very useful for the production of single-
stranded versions of cloned genes they do suffer from one
disadvantage. There is a limit to the size of DNA fragment that can
be cloned with an Ml3 vector, with 500bp generally being looked
on as the maximum capacity.
•To get around this problem a number of novel vectors (phagemids)
have been developed by combining a part of the M13 genome
with plasmid DNA.