These are extra chromosomal, self replicating and double stranded covalently closed circular DNA molecules present in the bacterial cell. A number of plasmids are present in bacterial cells that have sufficient genetic information for their replication. Even though they are not necessary for the survival of the bacteria they carry such necessary information as drug resistance, metal resistance, toxin production, nitrogen fixation etc. Naturally occurring plasmids can also be modified by in vitro techniques such as code transformation.
Use of plasmids as a vector for DNA cloning was reported for first time by Cohen et al (1973). Plasmids are used as cloning vehicles when they possess the following features. 1.
It can be readily isolated from the cells 2. It possesses a single restriction site for one or more restriction enzyme(s) 3. Insertion of a linear molecule at one of these sites does not alter its replication properties. 4. It can be reintroduced into a bacterial cell and cells carrying the plasmid with or without the insert can be selected or identified (Bernard and Helinski, 1980). 5. They do not occur free in nature but are found in bacterial cells. In many instances the main objective of coloning is the insertion of a particular fragment of DNA into a suitable plasmid vector and its amplification.
Amplification is a process of increasing the number of plasmids in a bacterial cell. In this process, a cell containing a relaxed plasmid is treated with a drug to inhibit protein synthesis. Consequently replication stops after the relaxed plasmid (eg pBR 322) continues to replicate despite the drug treatment. Addition of chloramphenicol causes the plasmid BR 322 to increase its number to 3,000 per cell. In order to replicate and clone a piece of foreign DNA, it is necessary to possess a sequence of nucleotides which is recognized by the host bacterium as origin of replication.
The origin of replication occurs naturally in plasmids. In naturally occurring plasmids due to exchange of genetic information between plasmids and bacterial chromosome new genes originate in the plasmids. Therefore plasmids have been artificially developed with specific sequence of genes so that they would integrate foreign DNA without any problem. The plasmid BR 322 is such an artificially constructed plasmid using two plasmids of E.coli – PBR 318 and PBR 322.
This plasmid contains the origin of replication (ori) derived from a plasmid related to naturally occurring plasmid Col El. As a result the BR 322 plasmid replicates at a rate faster than bacterial genome. It also possesses genes conferring resistance to antibiotics such as ampicillin and tetracyclin. It also has a unique recognition site for 20 restriction enzymes.
Certain restriction sites, for example Bam HI in the tet gene of the plasmid is present within the gene in such a way that the insertion of foreign DNA will inactivate the gene. As a result, the recombinant plasmid will allow the cells to grow only in the presence of ampicillin but will not protect them against tetracyclin. This antibiotic resistance will allow the selective isolation °f cells which can be cultured on a medium supplemented with ampicillin. In addition to the E.coli plasmids a plasmid of gram negative bacteria called RK2 plasmid has been developed which has a very broad host range and has a single restriction site which can be acted upon by several end nucleases.
Isolation of bacterial plasmids: The following steps are necessary for isolating the bacterial plasmids 1. Treat the prokaryotic cells with detergents which will release plasmid DNA and chromosome from the bacterial cell along with other molecules. This mixture is known as lysate.
2. Treat the lysate with potassium acetate or acetic acid solution. This precipitates chromosomal DNA and some protein molecules. 3. Remove the precipitate from the lysate by high speed centrifugation.
The supernatant contains plasmid DNA along with RNA, protein and some DNA debris of the chromosome. 4. Treat the lysate with RNAase, this will digest the RNA. 5. The lysate is treated with phenol and as a result two layers are formed – phenol layer and water layer.
The water layer contains plasmid DNA and the other contaminants are found in the phenol layer.
A virus which attacks the bacteria is called a bacteriophage.
A number of bacteriophages with different genetic materials have been reported so far from a variety of bacteria. Some of these phases are phages X, M13, Fdl 1, R209 etc. Phages also can be used as cloning vectors like plasmids. Of all the phages the X phage perhaps is the one that has been used very frequently as a cloning vector. The X phage contains a protein acetous head and a long tail. The head portion has 50 genes in its 49 kb genome of which about half of the genes are essential.
On attachment to the host (E.coli) the linear DNA molecule is injected into the cell. The linear double stranded molecule of DNA becomes a circle by joining through the single strand of 12 nucleotides commonly known as cos site. This cos site is a very important feature of X DNA The X phage has two kinds of life cycles – the lytic phase and the lysogenic phase in the bacterial cell. In the lytic pathway early in the infection the circular DNA replicates by a rolling circle mechanism and produces long molecules of DNA. At the same time the phage DNA directs the synthesis of proteins to produce the head.
In order to pack the DNA into the head it undergoes cleavage to yield the fragments of such sizes as to fit in the head. Eventually a tail is attached to the head. In the lysogenic pathway the phage genome integrates with the bacterial genome at an attachment site (att) where it replicates along with the bacterial genome.
The following are the advantage of using phage as cloning vectors – 1. DNA can be packed in vitro into phage particles and transduced into E.coli with high efficiency, 2. Foreign DNA up to 25 kb in length can be inserted into phage vector, and 3. Screening and storage of recombinant DNA is easier (Dahl et al.
, 1981). Before using the X phage vector it will be essential to remove the genome from the restriction sites for the enzymes commonly used for cloning. Two types of phage cloning vectors have been constructed – these are the insertion vector and the replacement vector.
Insertion vector these have unique cleavage sites into which a relatively small piece of foreign DNA can be inserted. These fragments do not affect the functioning of the phage. The upper and lower limit of DNA that may be packed into the phage particles is between 35-53 kb. The maximum size of a foreign DNA that can be inserted is about 18kb. Replacement vector these vectors have cleavage sites present on either side of the length of non essential DNA. As a result of cleavage left and right arms are formed and each arm has a terminal site and a longer stiffer region (the non essential region) which can be substituted by foreign DNA.
The maximum size of foreign DNA that can be inserted into this vector depends on how much the phage DNA is non essential. It has been estimated that about 25- 35kb of genome can be replaced with foreign DNA segments example of substituted vectors are-gt, WES and X 1059.
These may be defined as the hybrid vectors derived from the plasmids which Contain cos site of X phage. Reported for the first time by Colins and Hohn (1975) cosmids lack genes for encoding viral proteins Therefore neither viral particles are formed in the host cell nor does cell lysis occur. Special features of cosmids similar to plasmids are origin of replication, a marker gene coding for antibiotic resistance, a special cleavage site for the insertion of foreign DNA and the small cells. Characters of cosmids dissimilar to plasmids are presence of extraphage DNA, and the cos site.
When plasmids are inserted into the genome of X phage they are said to be phasmids. The process of insertion of the plasmid DNA into the phage genome occurs in the same way as a phage genome is inserted into the bacterial chromosome.
Insertion of plasmid into X phage is done with a view to have a specific site responsible for recombination insertion of the phage into bacterial chromosome. Phasmids may be used in many ways. They can be used as a phage cloning vector from which a recombinant vector may be released.