Biology FEATURES REQUIRED FOR CLONING IN A VECTOR

### KEY TOPICS

star Cloning Vectors
star Origin of replication
star Selectable marker
star Cloning sites
star Vectors for cloning genes in plants and animals

### CLONING VECTORS

● 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.

### ORIGIN OF REPLICATION (ori)

● 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.

### SELECTABLE MARKER

● 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.

### CLONING SITES

● 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".

### VECTORS FOR CLONING GENES IN PLANTS AND ANIMALS

● 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 thecolor{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).