`star` Insertion of Recombinant DNA into the Host Cell/Organism
`star` Obtaining the Foreign Gene Product
`star` Bioreactors
`star` Downstream Processing


● There are several methods of `color{Violet}"introducing the ligated DNA"` into recipient cells.

● Recipient cells after making them `color{Violet}"competent"` to receive, `color{Violet}"take up DNA"` present in its surrounding.

● So, if a recombinant DNA bearing `color{Violet}"gene for resistance"` to an antibiotic (e.g., ampicillin) is transferred into `color{Violet}"E. coli cells"`, the host cells become transformed into `color{Violet}"ampicillin-resistant cells"`.

● If we spread the transformed cells on `color{Violet}"agar plates"` containing `color{Violet}"ampicillin"`, only `color{Violet}"transformants will grow,"` untransformed recipient cells will die.

● Since, due to `color{Violet}"ampicillin resistance gene"`, one is able to `color{Violet}"select a transformed cell"` in the presence of ampicillin.

● The `color{Violet}"ampicillin resistance gene"` in this case is called a `color{Violet}"selectable marker"`.

Obtaining the Foreign Gene Product

● When a `color{Violet}"piece of alien DNA"` is inserted into a `color{Violet}"cloning vector"` and transferred it into a bacterial, plant or animal cell, the `color{Violet}"alien DNA gets multiplied"`.

● In almost all `color{Violet}"recominant technologies"`, the ultimate aim is to produce a `color{Violet}"desirable protein"`.

● Hence, there is a need for the `color{Violet}"recombinant DNA"` to be `color{Violet}"expressed"`.

● The foreign gene gets expressed `color{Violet}"under appropriate conditions"`.

● The `color{Violet}"expression of foreign genes"` in host cells involve understanding many `color{Violet}"technical details"`.

● After having `color{Violet}"cloned the gene"` of interest and having `color{Violet}"optimised the conditions"` to induce the expression of the target protein, one has to consider `color{Violet}"producing it on a large scale"`.

● If any `color{Violet}"protein encoding gene"` is expressed in a `color{Violet}"heterologous host"`, is called a `color{Brown}"recombinant protein"`.

● The cells harbouring `color{Violet}"cloned genes of interest"` may be grown on a small scale in the `color{Violet}"laboratory"`.

● The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.

● The cells can also be multiplied in a `color{Brown}"continuous culture system"` wherein the `color{Violet}"used medium"` is drained out from one side while `color{Violet}"fresh medium"` is added from the other to maintain the cells in their physiologically most active `color{Violet}"log/exponential phase"`.

● This type of culturing method produces a `color{Violet}"larger biomass"` leading to higher yields of `color{Violet}"desired protein"`.

`color{Violet}"Small volume cultures"` cannot yield appreciable quantities of products.


● To produce in `color{Violet}"large quantities"`, the development of `color{Brown}"bioreactors"`, where large volumes `color{Violet}"(100-1000 litres)"` of culture can be processed, was required.

● Thus, bioreactors can be thought of as `color{Violet}"vessels"` in which raw materials are `color{Violet}"biologically converted"` into specific products, individual enzymes, etc., using `color{Violet}"microbial plant, animal"` `color{Violet}"or human cells"`.

● A bioreactor provides the `color{Violet}"optimal conditions"` for achieving the `color{Violet}"desired product"` by providing `color{Violet}"optimum growth conditions"` (temperature, pH, substrate, salts, vitamins, oxygen).

● A `color{Violet}"stirred-tank reactor"` is usually cylindrical or with a curved base to `color{Violet}"facilitate the mixing"` of the reactor contents.

● The stirrer facilitates even `color{Violet}"mixing and oxygen availability"` throughout the bioreactor.

● Alternatively `color{Violet}"air can be bubbled"` through the reactor.

● The bioreactor has an `color{Violet}"agitator system"`, an `color{Violet}"oxygen delivery system"` and a `color{Violet}"foam control system"`, a `color{Violet}"temperature control system"`, `color{Violet}"pH control system"` and `color{Violet}"sampling ports"` so that small volumes of the culture can be withdrawn periodically.


● After completion of the `color{Violet}"biosynthetic stage"`, the product has to be subjected through a `color{Violet}"series of processes"` before it is ready for marketing as a `color{Violet}"finished product"`.

● The processes include `color{Violet}"separation and purification"`, which are collectively referred to as `color{Brown}"downstream processing"`.

● The product has to be formulated with `color{Violet}"suitable preservatives"`.

● Such formulation has to undergo thorough `color{Violet}"clinical trials"` as in case of `color{Violet}"drugs"`.

● `color{Violet}"Strict quality control"` testing for each product is also required.

● The `color{Violet}"downstream processing"` and quality control testing `color{Violet}"vary from product"` to product