Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophage

GENE CLONING AND DNA ANALYSIS

基因工程與原理

2 Vectors for Gene Cloning: Plasmids and Bacteriophages

2.1 Plasmids2.1.1 Size and copy number2.1.2 Conjugation and compatibility2.1.3 Plasmid classification2.1.4 Plasmids in organisms other than bacteria

2.2 Bacteriophages2.2.1 The phage infection cycle2.2.2 Lysogenic phagesGene organization in the DNA moleculeThe linear and circular forms of DNAM13—a filamentous phage2.2.3 Viruses as cloning vectors for other organisms

2.1 Plasmids

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

Figure 2.2The use of antibiotic resistance as a selectable marker for a plasmid. RP4 (top) carries genes for resistance to ampicillin, tetracycline and kanamycin. Only those E. coli cells that contain RP4 (or a related plasmid) are able to survive and grow in a medium that contains toxic amounts of one or more of these antibiotics.

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

origin of replication

2.1.1 Size and copy number

The factors that control copy number are not well understood.Stringent v.s. Relaxed

Figure 2.4Plasmid transfer by conjugation between bacterial cells. The donor and recipient cells attach to each other by a pilus, a hollow appendage present on the surface of the donor cell. A copy of the plasmid is then passed to the recipient cell. Transfer is thought to occur through the pilus, but this has not been proven and transfer by some other means (e.g. directly across the bacterial cell walls) remains a possibility.

2.1.2 Conjugation and compatibility Several different kinds of plasmid may be found in a single cell.

2.1.3 Plasmid classification

1. Fertility or F plasmids carry the tra genes.2. Resistance or R plasmid carry genes conferring on the host

bacterium resistance to one or more antibacterial agents.3. Col plasmids code for colicin, proteins that kill other bacteria.4. Degradative plasmids allow the host bacterium to metabloize

unusual molecules. (TOL of Pseudomonas putida 戀臭假單胞菌 )

5. Virulence plasmids confer pathogenicity on the host bacterium. (Ti plasmid of Agrobacterium tumefaciens農桿菌 )

2.1.4 Plasmids in organisms other than bacteria

The eukaryotic plasmid, 2 m circle, occurs in many strains of the yeast Saccharomyces cerevisiae.

Figure 2.5The two main types of phage structure: (a) head-and-tail (e.g. λ); (b) filamentous (e.g. M13).

2.2 Bacteriophages

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

2.2.1 The phage infection cycle

lytic cycle

Figure 2.7The lysogenic infection cycle of bacteriophage lambda (λ).

2.2.2 Lysogenic phages

The prophage is quiescent.

The bacterium referred as a lysogen.

Figure 2.8The infection cycle of bacteriophage M13.

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

Clustering of related genes/ switched on and off as a group

Figure 2.10The linear and circular forms of λ DNA. (a) The linear form, showing the left and right cohesive ends. (b) Base pairing between the cohesive ends results in the circular form of the molecule. (c) Rolling circle replication produces a catenane of new linear λ DNA molecules, which are individually packaged into phage heads as new λ particles are assembled.

Sticky ends or cohesive ends

1. Circularized/ insertion into bacterial genome.

2. Rolling circle replication

Figure 2.11The M13 infection cycle, showing the different types of DNA replication that occur. (a) After infection the single-stranded M13 DNA molecule is converted into the double-stranded replicative form (RF). (b) The RF replicates to produce multiple copies of itself. (c) Single-stranded molecules are synthesized by rolling circle replication and used in the assembly of new M13 particles.

RF: 100 copies/ cell

1000 new phages being produced during each generation of infected cell

Several features of M13 make this phage attractive as a cloning vector:1.Less than 10 kb in size.2.The double-stranded replicative form (RF)3.Can be obtained in the form of single-stranded DNA4.Useful for the DNA sequencing and in vitro mutagenesis.5.Phage display: identify the proteins interaction.

2.2.3 Viruses as cloning vectors for other organisms

Adenoviruses for humanBaculoviruses for insect cellsCaulimoviruses and geminviruses for plants.