● It is known that `color{Violet}"plasmids and bacteriophages"` have the ability to replicate within `color{Violet}"bacterial cells"` independent of the `color{Violet}"control of chromosomal DNA"`.

● Bacteriophages because of their `color{Violet}"high number per cell"`, have very `color{Violet}"high copy numbers"` of their genome within the bacterial cells.

● Some plasmids may have only `color{Violet}"one or two copies"` per cell whereas others may have `color{Violet}"15-100 copies"` per cell. Their numbers can go `color{Violet}"even higher"`.

● If we are able to link an `color{Violet}"alien piece of DNA"` with bacteriophage or plasmid DNA, we can `color{Violet}"multiply its numbers"` equal to the copy number of the `color{Violet}"plasmid or bacteriophage"`.

● Vectors used at present, are engineered in such way that they help `color{Violet}"easy linking of foreign DNA"` and selection of recombinants from non-recombinants.

● Many features that are required to `color{Violet}"facilitate cloning"` into a `color{Violet}"vector"` are taken into consideration.

● This is a sequence from where `color{Violet}"replication starts"` and any piece of DNA when linked to this sequence can be made to replicate within the host cells.

● This sequence is also responsible for `color{Violet}"controlling the copy number"` of the linked DNA.

● So if one wants to `color{Violet}"recover many copies"` of the target DNA it should be cloned in a vector whose `color{Violet}"origin"` support high copy number.

● In addition to ‘ori’, the vector requires a `color{Brown}"selectable marker,"` which helps in `color{Violet}"identifying and eliminating"` non transformants and selectively `color{Violet}"permitting the growth"` of the transformants.

● `color{Brown}"Transformation"` is a procedure through which a piece of DNA is introduced in a `color{Violet}"host bacterium"`.

● Normally, the genes encoding resistance to antibiotics such as `color{Violet}"ampicillin, chloramphenicol"`, `color{Violet}"tetracycline or kanamycin"`, etc., are considered useful selectable markers for E. coli.

● The `color{Violet}"normal 𝘌. 𝘤𝘰𝘭𝘪 cells"` do not carry resistance against any of these antibiotics.

● In order to link the `color{Violet}"alien DNA"`, the vector needs to have very few, `color{Violet}"preferably single, recognition sites"` for the commonly used restriction enzymes.

● Presence of `color{Violet}"more than one"` recognition sites within the vector will generate `color{Violet}"several fragments"`, which will complicate the gene cloning.

● The `color{Violet}"ligation of alien DNA"` is carried out at a `color{Violet}"restriction site"` present in one of the two antibiotic resistance genes.

● For example, you can ligate a `color{Violet}"foreign DNA at the Bam H I"` site of tetracycline resistance gene in the vector `color{Violet}"pBR322"`.

● The `color{Violet}"recombinant plasmids"` will lose `color{Violet}"tetracycline resistance"` due to insertion of foreign DNA but can still be selected out from `color{Violet}"non-recombinant ones"` by plating the transformants on ampicillin containing medium.

● The transformants growing on `color{Violet}"ampicillin"` containing medium are then transferred on a medium containing `color{Violet}"tetracycline"`.

● The recombinants will grow in `color{Violet}"ampicillin containing medium"` but not on that containing tetracycline.

● But, `color{Violet}"nonrecombinants"` will grow on the medium containing `color{Violet}"both the antibiotics"`.

● In this case, one antibiotic resistance gene helps in `color{Violet}"selecting the transformants"`, whereas the other antibiotic resistance `color{Violet}"selection of recombinants"`.

● Selection of recombinants due to `color{Violet}"inactivation of antibiotics"` is a `color{Violet}"cumbersome procedure"` because it requires `color{Violet}"simultaneous plating"` on two plates having different antibiotics.

● Therefore, `color{Violet}"alternative selectable markers"` have been developed which differentiate recombinants from non-recombinants on the basis of their ability to `color{Violet}"produce colour"` in the presence of a chromogenic substrate.

● In this, a recombinant DNA is inserted within the `color{Violet}"coding sequence"` of an enzyme, `color{Violet}"â-galactosidase"`.

● This results into `color{Violet}"inactivation of the enzyme"`, which is referred to as `color{Brown}"insertional inactivation"`.



● The presence of a `color{Violet}"chromogenic substrate"` gives `color{Violet}"blue"` coloured colonies if the plasmid in the bacteria does not have an insert.

● Presence of insert results into `color{Violet}"insertional inactivation"` of the â-galactosidase and the colonies do not produce any colour, these are identified as `color{Violet}"recombinant colonies"`.

● Scientists have learnt the lesson of `color{Violet}"transferring genes"` into plants and animals from bacteria and viruses which have known this for ages – how to `color{Violet}"deliver genes"` to transform `color{Violet}"eukaryotic cells"` and force them to do what the bacteria or viruses want.

● For example, `color{Violet}"𝘈𝘨𝘳𝘰𝘣𝘢𝘤𝘵𝘦𝘳𝘪𝘰𝘶𝘮 𝘵𝘶𝘮𝘪𝘧𝘢𝘤𝘪𝘦𝘯𝘴"`, a pathogen of several dicot plants is able to deliver a piece of DNA known as `color{Violet}"T-DNA"` to transform normal plant cells into a `color{Violet}"tumor"` and direct these tumor cells to produce the chemicals required by the pathogen.

● Similarly, `color{Violet}"retroviruses in animals"` have the ability to transform normal cells into `color{Violet}"cancerous cells"`.

● A better understanding of the`color{Violet}" art of delivering genes"` by pathogens in their eukaryotic hosts has generated knowledge to transform these `color{Violet}"tools of pathogens"` into `color{Violet}"useful vectors"` for delivering genes of interest to humans.

● The `color{Violet}"tumor inducing (Ti) plasmid"` of Agrobacterium tumifaciens has now been modified into a `color{Violet}"cloning vector"` which is no more pathogenic to the plants but is still able to `color{Violet}"use the mechanisms"` to deliver genes of our interest into a variety of plants.

● Similarly, `color{Violet}"retroviruses"` have also been `color{Violet}"disarmed"` and are now used to deliver desirable genes into animal cells.

● So, once a gene or a DNA fragment has been `color{Violet}"ligated into a suitable vector"` it is transferred into a bacterial, plant or animal host (where it multiplies).