PRESENTATION.

1. Chapter 2
Vectors for Gene Cloning: Plasmids and
Bacteriophage

2. Properties of vector
 Able to replicate within host cell.
 Relatively small in size ideally less than 10
kb
 Two kinds of such DNA molecule in bacterial
cells are:
Plasmid
Bacteriophage chromosome

3. Plasmid
 Circular DNA molecules (Fig:2.1)
 Independent existence
 One or more genes for some useful
characteristics, may be used as a selectable
marker e.g antibiotic resistance genes (Fig:2.2)
 Origin of replication, able to multiply
independently. (Fig:2.3a)
 May use host’s cell replicative enzymes
 Some are integrative plasmids (Fig:2.3b)

4. Figure 2.1. Plasmids: independent genetic
elements found in bacterial cells.

5. Figure 2.2. The use of antibiotic resistance as a
selectable marker for a plasmid.

6. Figure 2.3
Replication strategies for (a) a non-integrative plasmid,
and (b) an episome.

7. Size and Copy number
Plasmids range from 1 kb to 250 kb
(Table 2.1)
Copy number is the number of
molecules found in single bacterial cell.
Plasmids may be
Stringent- low copy number or
Relaxed- multiple copies (50 or more).

8. Conjugation and Compatibility
Plasmids falls in to two groups:
Conjugative- promote conjugation
between bacterial cells, have set of
transfer or tra genes (fig)
Non-conjugative
Different plasmids in a cell must be
compatible.
Incompatibility groups

9. Figure 2.4
Plasmid transfer by conjugation between bacterial cells.

10. Plasmid Classification
Based on the main characteristic coded by the plasmid genes
Five major types of naturally occurring plasmids
Fertility or F plasmids
 Only tra genes, promote conjugal transfer
Resistance or R plasmids
Resistance to antibacterial agents- RP4 Pseudomonas
Col plasmids
 Code for colicins (proteins that kill other bacteria)
Degradative plasmids (metabolize toluene and salicylic acid)
 Metabolize unusual compounds, TOL of Pseudomonas putida
Virulence plasmids
 Confers pathogenecity on the host, Ti of Agrobacterium tum.

11. Plasmids in organisms other than Bacteria
 Eukaryotic plasmid - 2µm circle- in many
strains of the yeast Saccharomyces
cerevisiae.
 Search for plasmids in other eukaryotes is
disappointing.

12. Figure 2.5
The two main types of phage structure: (a) head-and-
tail (e.g. λ); (b) filamentous (e.g. M13).
Viruses that specifically infect bacteria
Simple in structure , DNA (or RNA) and Capsid

13. The Phage infection cycle- Lytic cycle
 Pattern of infection is a three-step process
(fig 2.6)
1. Attachment of phage and injection of DNA
2. Replication
3. New phage particles assembly and
released causing lysis of cell
Completes within 20 minutes

14. Figure 2.6
The general pattern of infection of a bacterial cell by a
bacteriophage.

15. Lysogenic Phages
 Insertion of phage DNA into bacterial genome
(prophage)
 Retention of the phage in the host, for many
cell divisions
 Eventually released and reverts to lytic mode
 Many lysogenic phages but two have major
role in gene manip.
 λ – a typical lysogenic phage
 M13- a lysogenic phage, infects E.coli but is
not integrated , just slow the rate of growth
and division

16. The lysogenic infection cycle of bacteriophage λ.

17. Gene organization in the λ DNA molecule
 λ – typical phage
 49 kb in size
 Position and identities of all the genes are
known (fig 2.9)
 All functional genes are clustered together in the
genome- important for controlling expression of
the λ genome and construction of vector.
 it allows genes to be switched on and off as a
group rather than individually

18. Figure 2.9
The λ genetic map, showing the positions of the important
genes and the functions of the gene clusters.

19. Linear and circular forms of λDNA
 λDNA - linear double stranded
 “Sticky” ends (cos sites) form circular molecule
(fig 2.10a and fig 2.10b) and do rolling circle
(fig 2.10c)
 cos sites- recognition sequences for
endonuclease (product of gene A).
 Cleavage and packaging recognize just cos
sites and DNA sequence to either sides.

20. Figure 2.10
The linear and circular forms of λ DNA.

21. M13- a filamentous phage
 Filamentous phage, 6407 nucleotides single stranded
circular DNA.
 Genome is less than 10 kb in size
 Only 3 genes for capsid construction, 15 for λ
 Simpler infection cycle
 Infection into E.coli via pilus
 ssDNA act as template for synthesis of dsDNA
 Replicate over 100 copies
 Double stranded replicative form (RF)
 Cloned genes can be obtained in the form of ssDNA
 New phage particles are continuously assembled and
released (fig 2.11c)

22. The infection cycle of bacteriophage M13.

23. Figure 2.11
The M13 infection cycle, showing the different types of
DNA replication that occur.

24. Viruses as Cloning Vectors for other
organisms
For example
 Adenoviruses in gene therapy
 Baculoviruses to synthesize important
pharmaceutical proteins in insect cells
 Caulimoviruses and geminiviruses for cloning
in plants