*2.2.4 Cosmids*

*Genetic Engineering - A Glimpse of Techniques and Applications*

they still possess intact tetracycline-resistant genes.

The plasmids can be classified majorly into two categories:

copy number.

*2.2.2 Plasmids*

(**Figure 1**).

*2.2.3 Phage vectors*

bactericide production, etc. [13].

kinds are not generally employed as vectors.

extensively used in genetic engineering as vectors [14].

number of the gene of interest. If a large number of copies of DNA of interest are required, it must be cloned in a vector containing ori site supporting high

• **Cloning site**—It is site that is recognized by the restriction enzyme (preferable one) so that the gene of interest maybe inserted after digestion of the plasmid. The presence of more than a single cloning site may complicate the process of gene cloning as multiple fragments will be generated. The insertion of gene of interest is done at a restriction site present among two antibiotic resistance genes. For example, in the vector pBR322, a foreign piece of DNA can be joined at BamH I site of tetracycline resistance. Due to this, the recombinant might lose tetracycline resistance as its sequence is altered by insertion of foreign DNA, and thus the plasmids will lose tetracycline resistance. The transformants can still be distinguished from non-transformed ones by plating them on ampicillin medium. The transformed ones can grow on ampicillin medium but when transferred to a medium containing tetracycline, they are unable to grow, whereas the non-transformants can still grow on tetracycline medium as

• **Selectable marker**—Within a vector a selectable marker plays a key role in the identification of transformed cells from the non-transformed ones. Usually a selectable marker provides resistance towards antibiotics which are used for selection of transformants from non-transformants. These markers can provide resistance towards antibiotics like ampicillin, tetracycline, etc.

These are extrachromosomal DNA that possess the ability to replicate independently within the host cell. Plasmids are circular in shape and impart special properties to the organism possessing it as it may code for antibiotic resistance,

The first type is self-transmissible, i.e., the ones that possess the ability to promote conjugation and are transferred quickly amid the bacterial population. These

The second type is the non-self-transmissible, i.e., the ones that cannot regulate their own transfer among bacterial population. These categories of plasmids are

The gene of interest or the foreign gene is inserted within the plasmid DNA vector; for that the closed circular plasmid is cleaved using restriction enzyme to make it linear. A plasmid contains a single cleavage site for a restriction enzyme to avoid multiple digestions. The gene of interest is inserted in the linear plasmid, and then the recombinant is converted to its original circular form [15]

The viruses that infect the bacteria are termed as bacteriophages. They possess a very simple structure consisting of a genetic material which might be a DNA or RNA surrounded by a protein coat termed as capsid. These phages can also be employed as a vector as large piece of DNA of interest can be incorporated in the genetic material of the phage. Different phages have been developed for the

**4**

A cosmid is basically a plasmid with cos site, which contains cos sequences necessary for packaging. Cos sequences are ~200 base pair in length. Cosmids are hybrids comprising phage DNA molecule and plasmid of bacteria. Just like any other vector system, cosmids also require a selectable marker and an ori site. The cosmids lack λ-genes so unlike phages they do not produce plaques. Instead plaques colonies are formed by using selective media according to the marker just as done in the case of plasmids (**Table 1**).

## **2.3 Introducing vector into host cells**

The membrane of the cell prevents the DNA from diffusing in or out. This barrier must be manipulated or altered so that the host cells are able to take up the foreign DNA. This goal can be achieved by different ways.


**Table 1.**

*Enzymes involved in genetic engineering and their function.*
