CaCl₂ is known to increase the efficiency of DNA uptake to produce transformed bacterial cells. The divalent Ca²⁺ ions create transient pores on the bacterial cell wall by which the entry of foreign DNA is facilitated into the bacterial cells.

Restriction nuclease also called restriction enzymes (molecular scissors) are used for cutting of DNA at a specific location. These are the basic tool of biotechnology

Restriction endonuclease, EcoRI cuts the DNA strands a little away from the centre of the palindromic, sequence, but between the same two bases on the two strands.
\({ 5 }^{ ' }-\begin{matrix} { G }^{ \downarrow } & A \end{matrix}\begin{matrix} A & T \end{matrix}\begin{matrix} T & C \end{matrix}-{ 3 }^{ ' }\)
\({ 3 }^{ ' }-\begin{matrix} { C } & T \end{matrix}\begin{matrix} T & A \end{matrix}\begin{matrix} { A }_{ \uparrow } & G \end{matrix}-{ 5 }^{ ' }\)
This leaves single-stranded portions, called sticky ends, overhanging at the end of each strand.
Since, the stickiness facilitates the action of DNA ligase, they easily form hydrogen bonds with their complementary counterparts.

(i) Microinjection
(ii) Biolistics/ gene gun
(iii) Heat-Shock method
(iv) Using  disarmed pathogen vectors.

(i) Exonucleases cleave base pairs of DNA at their terminal ends (either 5' or 3') while, the endonucleases cleave DNA at any point within the DNA segment at specific position except terminal ends.
(ii) Restriction endonuclease EcoRI cuts the DNA strands a little away from the palindromic sequences, but between the same two bases on the two strands
5' - G - A - A - T - T - C - 3'
3' - C - T - T - A - A - G - 5'

Penicillin is a secondary metabolite and secondary metabolites are produced only when the organism is under stress, i.e, due to shortage of nutrients or environmental stress, etc. In continuous culture, the nutrients are added at continuous intervals and it remains in exponential phase, therefore less penicillin is produced (organism is never under stress). This is why batch culture (nutrients are added once) rather than continous culture is used for production of penicillin.

Application These bioreactors are used to produce large quantities of products enzymes, etc., using microbial, plant, animal of human cells.

Viroids are smallest and simplest pathogenic agents comprising of 300-4000 bases long, circular, single standed, naked RNA. They are mechanically transferable and infect other parts of the plants. They have capability of being used as plant vectors

Polymerase Chain Reaction
Multiple copies of the desired gene or segment of DNA, can be synthesised in vitro using two sets of primers, the oligonucleotides that are complementary to the regions of DNA of the two strands and the enzyme DNA polymerase.
This enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as the template.
For repeated amplification to be achieved, a thermostable DNA polymerase (Taq polymerase), extracted from the bacterium, Thermus aquaticus is employed; it remains active during the high temperature treatment used for denaturation and separation of the two strands.

Small volume cultures cannot yield appreciable quantities of products. To produce these products in large quantities the development of 'bioreactors' was required where large volumes (100-1000 litres) of culture can be processed. Thus bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, using microbial, plant, animal or human cells or individual enzymes.
Role. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). One of the most commonly used bioreactor is of stirring type. A stirred tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled 1lhroughthe reactor. If you look at the figure closely you will see that the bioreactor has an agitator system; an oxygen delivery system, a foam control system, a temperature control system, pH control system, sampling ports so that small volumes of the culture can be withdrawn periodically.

Small volume cultures cannot yield appreciable quantities of products. To produce these products in large quantities the development of 'bioreactors' was required where large volumes (100-1000 litres) of culture can be processed. Thus bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, using microbial, plant, animal or human cells or individual enzymes.
Role. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). One of the most commonly used bioreactor is of stirring type. A stirred tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled 1lhroughthe reactor. If you look at the figure closely you will see that the bioreactor has an agitator system; an oxygen delivery system, a foam control system, a temperature control system, pH control system, sampling ports so that small volumes of the culture can be withdrawn periodically.