Vectors Used for Cloning of Larger Molecules of DNA

In this article we will discuss about the three main types of vectors used in cloning for larger molecules of DNA. The types of vectors are: 1. Plasmid Vectors 2. Bacteriophage Vectors 3. Cosmid Vectors.

1. Plasmid Vectors:

Plasmids are the extra chromosomal genetic elements commonly found in bacteria and are mostly made of double-stranded circular DNA. They are used as vectors in gene cloning, because they have a replication origin in their DNA making them competent of autonomous replication, and also because they generally have one or two restriction sites for several restriction enzymes.

Many plasmids have been genetically engineered to add useful properties. The number of copies of plasmids may vary from one to several per host cell. Some plasmids under relaxed replication control can have larger number of copies which may be sometimes as high as 1,000. Such high copy-number plasmids are specially suitable for cloning.

A commonly used small cloning vector is the plasmid pBR 322 which has a circular double- stranded DNA having 4,363 base pairs, compared to the E. coli chromosome having about 4,700 x 10³ base pairs. It carries single restriction sites for the restriction enzymes EcoR1, Hind III, Bam HI and Sal I. Thus, each of these enzymes can make single cleavages at their respective sites of pBR 322 DNA, where the foreign DNA can be inserted.

Many plasmids also have genes for antibiotic resistance which can be profitably used as selectable markers. For example, pBR 322 has two genes specifying resistance to ampicillin (amp^r) and tetracycline (tet^r). Presence of two such marker genes is more advantageous than a single marker, provided one of the marker genes has a restriction site within it. When a foreign DNA fragment is inserted within the resistance gene, it results in the inactivation of the resistance gene, just as a transposon causes inactivation of a gene.

Such inactivation of an antibiotic resistance gene makes the host ceil in which the plasmid is present susceptible to the particular antibiotic. This property can be utilized for identification and selection of the host cells in which the foreign gene has been cloned. For example, pBR 322 possesses a restriction site for Hind III in the tet^r gene.

When a foreign gene is cloned using Hind III in pBR 322, tet^r gene is inactivated and the host cells containing the recombinant plasmid show resistance to ampicillin because the amp’ gene is intact, but not to tetracycline. This makes possible the identification of the host cells containing the cloned foreign gene, because they cannot grow on a medium in which tetracycline has been incorporated at an inhibitory concentration. Other host cells which take up only the vector DNA without the cloned gene will grow in such a medium, because their tet^r gene is intact.

Naturally occurring plasmids do not usually possess all the desirable properties which are useful for their use as cloning vectors. So they must be suitably altered by molecular biological techniques. For example, the plasmid pBR 322 was developed by several alternations. Other plasmids which are used as cloning vectors include pSC 101, pUC 8, pHC 79 etc. (p stands for plasmid).

A very useful plasmid used as a vector for introducing foreign genes into plants is the Ti-plasmid of a plant pathogenic bacterium, Agrobacterium tumefaciens which infects many dicotyledonous plants causing a disease, known as crown-gall. When this organism infects a host plant, it transfers naturally a portion of the Ti-plasmid into the plant. This portion is known T-DNA. The T-DNA segment has been extensively altered by molecular biological methods to make the Ti-plasmid suitable as a vector for inserting foreign DNA into many plants to produce transgenic plants.

2. Bacteriophage Vectors:

The most commonly used bacteriophage vector is the λ-(lambda) phage of E. coli.The λ-DNA after it infects a host cell can either enter into a lysogenic state by integration into the host DNA, or can lead to a lytic cycle producing progeny phages. In the phage head, the DNA is present as a linear double-stranded helix.

After entering into the host cell, the molecule forms a covalently closed circular DNA with the help of single-stranded ends having complimentary base sequences (cos-sites). The λ-genome is 48.5 x 10³ base-pair long and has a single restriction site for Eco R1. To be used as a cloning vector, the λ-DNA requires to be processed by genetical engineering techniques, so that it retains the genes necessary for carrying out the lytic cycle only and the Eco R1 recognition sequence.

The central portion of the genome which is not essential for the lytic cycle is removed to accommodate the inserted foreign DNA into λ-DNA, so that the recombinant DNA has the appropriate length to be packaged into the phage head.

One problem with the phage vectors is the length of the foreign DNA that is to be inserted. If the length of the insert is too long or too short, the recombinant DNA cannot be packaged into the phage head. The insert in case of λ-DNA should be about 10 to 20 kb, preferably 15 kb long. After the λ-DNA is processed, its length becomes too short to be packaged into the phage head.

The insertion of a foreign DNA having an appropriate length (-15 kb) brings back the length to an appropriate size which can be packaged into a λ -head. The recombinant phage can then be used for propagation in E. coli yielding large number of phage particles which carry the recombinant DNA.

The insertion of a foreign DNA fragment of appropriate length which carries a gene of choice is carried out by treatment of both insert and processed λ-DNA with Eco R1. The foreign DNA, with its sticky-ends, forms base pairs with those of λ-DNA fragments to yield a recombinant DNA. The recombinant molecule is then packaged into a phage head. The phage on infection produces a circular DNA and multiplies producing a lytic cycle. From the progeny phage particles, recombinant DNA can be isolated (Fig. 9.131).

For cloning larger DNA molecules, i.e. larger than 20 kb, the bacteriophage PI of E. coli can be used. This phage is also a temperate one like λ-phage, but it has a DNA genome of about 100 kb, i.e. double of λ-phage. Of its 100 kb genome, only about 15 kb is essential for replication in the host. So, a processed vector prepared from phage PI can be used for cloning much larger DNA fragments of 80 to 85 kb. The phage PI DNA is a circular molecule. The general procedure of inserting a foreign DNA into P1 vector is similar to that of λ -DNA.

3. Cosmid Vectors:

Cosmids are hybrids of plasmids and λ -phage. The cos-sites of λ-DNA are joined to a plasmid, like Col El, to produce a typical cosmid. A cosmid is generally a circular DNA molecule containing its own replication origin sequence, a selectable marker gene — like one coding for antibiotic resistance — one or two restriction sites and the cos-site of X-DNA. The cos-site helps in circularization of the cosmid DNA. The cosmid derived from Col El has a rifampicin-resistance marker (rif^r), two restriction sites for Hind III and the cos-site of λ-phage (Fig. 9.132).

Cosmids are suitable for cloning larger molecules of DNA. On the average, DNA fragments having a size of 35 to 45 kb can be inserted into a cosmid, whereas, in plasmids, the insert size is between 5 kb to 10 kb, and, in λ-vectors, the insert size is usually about 15 kb.