Cloning Vectors

To act as cloning vector, a DNA molecule must be capable of entering a host and replicate itself inside the host. In addition, it must ideally be small in size (less than 10 kb) as larger molecules tend to break down during purification and are difficult to manipulate. Two naturally occurring types of DNA molecule satisfy these requirements: Plasmids and Viral Chromosomes.

Plasmids

Plasmids are small circular DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome. Plasmids can carry one ore more genes. Most plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell independently of the chromosome.

A few types of plasmids insert themselves into the bacterial chromosome for replication. Such plasmids are called episomes.

The size and copy number of a plasmid is important. Copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell. Plasmids range from 1 kb to 250 kb. Plasmid smaller than 10 kb are preferred for vectors.

Plasmids can be conjugative or non-conjugative. Conjugative plasmids are characterized by their ability to promote sexual conjugation between bacterial cells. Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact. Conjugation and plasmid transfer are controlled by a set of transfer or tra genes which are present in conjugative genes but absent in non-conjugative genes.

To coexist in a cell, different plasmids must be compatible. If two plasmids are incompatible, one of them would quickly be lost from the cell. Thus plasmid can be assigned to different incompatibility groups. Naturally occuring plasmids are classified on the basis of the main characteristic code by the plasmid genes.

  1. Fertility or F plasmids: carry only tra genes and have no characteristic beyond the ability to promote conjugation
  2. Resistance or R plasmids: carry antibiotic resistance genes. Used as selectors in the lab
  3. Col plasmids: code for colicins which are proteins that kill other bacteria
  4. Degradative plasmids: allows the host bacteria to metabolize unusual molecules
  5. Virulence plasmids: confer pathogenicity on the host bacteria

Bacteriophages

Bacteriophages or phages are viruses that specifically infect bacteria. Viral chromosomes and chromosomes of bacteriophages, in particular, are ideal for inserting DNA into the host chromosome where they are replicated by the host chromosome.

Phages are very simple in structure. They consist DNA surrounded by a protective coat or capsid made up of proteins.

The general pattern of infection is a three-step process:

1. The phage attaches to the cell membrane of a bacteria and injects its DNA into the cell.
2. The inserted DNA is replicated by the bacteria's cell machinery
3. Protein components of the phage are assembled and the phages are released from the host

Lytic Cycle
If the cell infection cycle is very rapid (completed within minutes), it is called a lytic cycle as the release of phage particles is associated with lysis of bacterial cell. DNA is replicated immediately after insertion followed by capsid synthesis. The phage DNA is never maintained in a stable condition in the host cell.

Lysogenic Phages

Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are not transcribed; the phage genome exists in a repressed state. The phage DNA in this repressed state is called a prophage because it is not a phage but it has the potential to produce phage. In most cases the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen. [3]

λ phage is a typical lysogenic phage. In an M13 phage, new phage particles are continuously assembled and released from the cell. The phage DNA is neither quiescent for integrated in the bacterial chromosome. Cell lysis never occurs. Both λ and M13 have found major roles as cloning vectors.

Properties of λ DNA
In λ DNA, genes coding for functionally related proteins (such as capsid proteins) are clustered together. The linear molecule has a 12 nucleotide single strand stretch which is complementary to the other end. Thus the molecule can become circular. Such complementary ends are called cohesive ends or cos sites. Cos sites allows the DNA to be inserted into the bacterial genome, circularization is a necessary prerequisite. Cos sites also act as recognition sequences for endonucleases that cleave DNA at cos site, producing individual λ genomes.

M13
M13 is filamentous has a completely different structure from λ. M13 genome is much smaller than λ genome, circular and single stranded. M13 DNA is inserted into E. coli during sexual conjugation via a pilus. Once inside the DNA is complemented and replicated. The DNA is never inserted in the bacterial genome.

The small size of M13 genome is very desirable. The double-stranded replicative form (RF) of M13 genome behaves like a plasmid and can be treated as such for experimental purposes. It is easily prepared and reintroduced in the cell by transfection. Using M13 vector is an easy and reliable way of obtaining single-stranded DNA.

Sources

[1] Gene cloning and DNA analysis by T. A. Brown
[2] http://en.wikipedia.org/wiki/Bacterial_conjugation
[3] http://pathmicro.med.sc.edu/mayer/phage.htm

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