Biotechnology Principles and Processes

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

According to European Federation of Biotechnology ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

1.0Principles of Biotechnology

Two core techniques

1. Genetic Engineeering : Techniques to alter the chemistry of genetic material (DNA and RNA).
2. Bioprocess Engineering: Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes.

2.0Advantages of Genetic Engineering over Traditional Hybridization

Traditional hybridization - lead to inclusion and multiplication of undesirable genes along with the desired genes. 

genetic engineering - allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism. 

Three Basic Steps of Genetic Engineering

(i) Identification of DNA with desirable genes;

(ii) Introduction of the identified DNA into the host;

(iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

History of Genetic Engineering

In 1972, Stanely Cohen and Herbert Boyer constructed first recombinant molecule.

Diagrammatic representation of recombinant DNA technology

History of Restriction Enzymes:

• In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. 
• One of these added methyl groups to DNA, while the other cut DNA.
• The first restriction endonuclease Hind II.
• Today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria

Nomenclature of Restriction Enzymes:

• It consist of 3/4 letter + one Roman number.
• The first letter :- Indicates  genus of bacteria.
• Second and third letter : Indicates species of bacteria.
• Fourth letter : Indicates strain of bacteria (optional).
• Roman numerical :  Order in which the enzymes were isolated from that bacteria.

Recognition Sequence:

• Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
• The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. For example, 

                                5' —— GAATTC —— 3'

                                 3' —— CTTAAG —— 5'

How Do Restriction Enzymes Work :

• Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence.
• Once it finds its specific recognition sequence,
• It will bind to the DNA 
• cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones.

Sticky Ends:

• Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. 
• This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand.
• These are named so because they form hydrogen bonds with their complementary cut counterparts.

Other Enzymes:

• Joining enzyme – The same kind of ‘sticky-ends’ can be joined together (end-to-end) using DNA ligases (Molecular glue).
• Synthesizing enzymes – DNA polymerase Synthesizes DNA from DNA.

3.0Vectors

Vectors used at present, are engineered in such a way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.

Features of a Cloning Vector:

(1) Origin of replication (ori) : 

• This is a sequence from where replication starts.
• Any piece of DNA when linked to this sequence can be made to replicate within the host cells. 
• This sequence is also responsible for controlling the copy number of the linked DNA.

(2) Selectable marker : 

• It helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. 
• The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli. 
• The normal E. coli cells do not carry resistance against any of these antibiotics.
• Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.

(3) Cloning sites: 

• In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. 
• Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning. 
• The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

Types of Vector

(1) Plasmid Vector : 

• Plasmids are extra-chromosomal and autonomously replicating circular ds DNA.

• E. coli cloning vector pBR322 showing :
• Restriction sites (Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I), 
• Ori 
• Antibiotic resistance genes (ampR and tetR). 
• Rop codes for the proteins involved in the replication of the plasmid.

(2) Disarmed Vectors :

(a) Agrobacterium tumifaciens for Plant cells : 

• Agrobacterium tumifaciens, a pathogen of several dicot plants. 
• It is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
• The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to the plants.
• It is still able to use the mechanisms to deliver genes of our interest into a variety of plants.

(b) Retroviruses for Animal cells  :

• Retroviruses have the ability to transform normal cells into cancerous cells.
• Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.

Competent Host (For Transformation with Recombinant DNA)

DNA is a hydrophilic molecule, it cannot pass through cell membranes.

Bacterial cells → treat with divalent cation Ca²⁺ → Incubate cells with rDNA on ice

↓ 

Bacteria takes up rDNA ← Put them on ice ← Place at 42°C (heat shock)

4.0Other Methods of Transformations

This is not the only way to introduce alien DNA into host cells. In a method known as :

1. Micro-injection :

Recombinant DNA is directly injected into the nucleus of an animal cell. 

2. Biolistics or gene gun : 

• It is suitable for plants cells. 
• Plant cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA 

5.0Processes of Recombinant DNA Technology

(I) Isolation of the Genetic Material (DNA)

Nucleic acid is the genetic material of all organisms without exception.

Step-I Lysis of cell : Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA.

Step-II Removal of other macromolecules :

RNA- removed by Ribonuclease

Protein - removed by Protease

Step-III Precipitation of DNA : Addition of chilled ethanol precipitates DNA.

Step-IV Spooling of DNA : Collection fine threads of DNA using glass rod.

(II) Fragmentation Of DNA By Restriction Endonucleases

Separation and isolation of DNA fragments :

• The cutting of DNA by restriction endonucleases results in the fragments of DNA. 
• DNA fragments can be separated by a technique known as gel electrophoresis. 
• Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. 
• Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds.
• The sieving effect is provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.

(III) Amplification of Gene of Interest Using PCR

PCR - Polymerase Chain Reaction.

• Multiple copies of the gene (or DNA) of interest are synthesised in vitro.
• Reaction mixture for PCR.

• Each cycle of PCR has three steps : 

(IV) Ligation of the DNA Fragment Into a Vector

(V) Transferring the Recombinant DNA into the Host.

(VI) Selection of Transformant with Recombinant Cell

(a) Selection of recombinant on the basis to antibiotic to genes

(b) Selection of recombinant on the basis of insertional inactivation of β-galactosidase gene.

X-gal chromogenic substrate $\stackrel{\beta -galactosidaseEnzyme}{\to }$ Blue-coloured product. 

(VII) Obtaining the Foreign Gene Product

• If any protein-encoding gene is expressed in a heterologous host, it is called a recombinant protein.
• The expression of recombinant protein required optimal conditions.

A culture of cells harbouring cloned genes of interest can be grown.

• The most commonly used bioreactors are of stirring type.

Features of stirred-tank bioreactors are :

– Cylindrical or with a curved base: facilitate the mixing of the reactor contents. 

– Stirrer: even mixing and oxygen availability throughout the bioreactor. 

– Agitator system 

– Oxygen delivery system

– Foam control system 

– Temperature control system

– pH control system

– Sampling ports: Small volumes of the culture can be withdrawn periodically.

Continuous Culture System:

• The used medium is drained out from one side while the fresh medium is added from the other.
• The cells are maintained in their physiologically most active log/exponential phase. 
• It produces a larger biomass leading to higher yields of desired protein.

(VIII) Downstream Processing

• It includes the separation and purification of the desired product (recombinant protein).
• The product has to be formulated with suitable preservatives and undergo thorough clinical trials. 
• Strict quality control testing for each product (which varies from product to product).