BIOTECHNOLOGY- PRINCIPLES AND PROCESSES

BIOTECHNOLOGY - PRINCIPLES & PROCESSES

Biotechnology term given by Karl Ereky. Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and process useful to humans.

 Types of Biotechnology Two types- 

(1) Old/traditional- 

Old biotechnology are based on the natural capabilities of micro organisms.

e.g. formation of Citric acid, production of penicillin by Penicillium notatum, making curd bread or wine, which are all microbe mediated processes a form of biotechnology.

(2) New/modern-

Based on Recombinant DNA technology.

e.g Human gene producing Insulin has been transferred and expressed in bacteria like E.coli.

 According to the European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modem molecular biotechnology. The definition given by EFB is as  follows: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Paul berg (Father of genetic engineering)-  He transferred gene of SV-40 virus (simian virus) in to E.coli with the help of  -phage (Nobel prize 1980).

Stanley Cohen and Herbert Bover: First made recombinant DNA by linking an  antibiotic resistance gene with a native plasmid of Salmonella typhimurium.

Principles of Biotechnology 

Among many, the two core techniques that enabled birth of modern biotechnology are:-

1. Genetic Engineering/Recombinant DNA Technology:

 Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. (i.e. formation  of genetically modified organism).

2. Bioprocess Technology:

Maintenance of sterile (microbial contamination free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture biotechnological products like antibiotics, vaccines.enzymen, etc.

The concept of genetic engineering was the outcome of two very significant discoveries made in bacterial research. These were-

1. presence of extrachromosomal DNA fragments called plasmids in the bacterial cell, which replicate along with chromosomal DNA of the bacterium.

2. Presence of enzymes restriction endonucleases which cut DNA at specific sites. These enzymes are, Therefore, called 'molecular scissors'.

The main basis of Recombinant DNA Technology is DNA cloning:- it is  making multiple identical copies of any template DNA.

There are three basic steps of DNA clonning-

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.

TOOLS OF RECOMBINANT DNA TECHNOLOGY :

Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to produce a recombinant DNA (rDNA). The 'biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA, desired DNA and host cells.

1. Enzymes :- A number of specific kinds of enzymes are employed in genetic engineering.

These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes.

Lysing enzymes - These enzymes are used for opening the cells to get DNA for genetic experiment.

Bacterial cell : is commonly digested with the help of lysozyme.

Plant cell : is commonly digested with the help of cellulase and pectinase

• Fungal cell : is commonly digested with the help of chitinase.

Cleaving enzymes - These enzymes are used for DNA molecules. Cleaving enzymes are of two types: exonucleases and endonucleases.

Exonuclease remove nucleotides from the ends of the DNA.

Endonucleases make cuts at specific positions within the DNA.

eg. Restriction endonucleases

Restriction Endonuclease Enzymes (Molecular scissors or molecular knife) Bestriction enzymes belong to a larger class of enzymes called endonucleases.

Restriction enzymes are used in recombinant DNA Technology because they can be used in vitro to recognize and cleave within specefic DNA sequence typically consisting of 4 to 8 nucleotides. This specie 4 to 8 nucleotide sequence  is called restriction site and is usually palindromic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.

Restriction enzymes are obtained from bacteria.

 What is function of restriction enzymes in bacteria?

They are useful to bacteria because the enzyme bring about fragmentation of viral DNA without affecting the bacterial genome. This is an adaptation against bacteriophages. These enzymes exist in many bacteria beside cleavage some restriction on endonuclease, also have capability of modification.

Modification in the form of methylation, by methylation the bacterial DNA modifies and therefore protects its an own chromosomal DNA from cleavage by these restriction enzymes.

 The first restriction endonuclease-Hind II, whose functioning depended on a specific DNA nucleotide sequence was  isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II.

Restriction enzyme (Eco R-I) was discovered by Arter Smith & Nathans (1978 Nobel prize).

Besides Hind II,  today we know more thán 900 restriction enzyme that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Nomenclature of enzyme -

The first letter- indicates bacterial genus (In italic).

 Second and third letter- indicate species of bacteria (in italics).

 Fourth letter- indicates strain of bacteria (optional).

 Roman numeraical- signifying the order in which the enzymes were Isolated from that strain of bacteria.

eg. EcoRI comes from Escherichia coli RY 13. In EcoRL the letter 'R' is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Restriction enzymes forms two types of ends on the basis of mode of cutting.

Sticky end (Free end) : 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 portion at the ends. There are overhanging stretches called sticky ends on each. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

eg. EcoRI, Hind III, Bam HI, Sal l.

Blunt end (non sticky end) : some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in blunt end or flush end.

eg. Sma l, EcoRV, Hae III.

 Sma l (Serratia marcescens)

Synthesizing enzymes- These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types; reverse transcriptases and DNA polymerases.

Reverse transcriptases help in the synthesis of complementary DNA strands on RNA templates.

DNA polymerases- help in the synthesis of complementary DNA strands on DNA templates. 

Joining enzymes- These enzymes help in joining the DNA fragments. For example DNA ligase from Escherichia coli is used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.

2. Vehicle DNA or Vector DNA-  The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle or vector DNA.

You know that plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

The following are the features that are required to facilitate cloning into a vector.

A. Origin of replication (ori) : This is a sequence from where 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 controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

B. Selectable marker: In addition to 'ori', the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

• Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc. are considered useful selectable markers for E.coli

• Enzyme forming gene also act as selectable marker gene, eg Lac. Z gene Note: The normal E. coli cells do not carry resistance against any of these antibiotics.

C. Restriction sites/Cloning sites : In order to link the alien DNA, the vector needs, recognition sites for the commonly used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

A vector should have restriction site for many enzyme but only one restriction site for each enzyme, Otherwise vector will get fragmented.

Selection of vector depends on :-

1. Size of desired gene 

2. Type of host 

Some examples of vectors:-

a. Plasmids- They are double stranded, circular and extra chromosomal DNA segments found in bacteria which can replicate independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments by means of restriction enzymes and DNA ligase. A plasmid carrying DNA of another organism integrated with it, is known as recombinant plasmid or hybrid plasmid or Chimeric plasmid.

eg. pBR 322, PUC 18 (used for gene transfer in bacteria).

Ti plasmid, Ri Plasmid (used for gene transfer in dicot plant).

b. Viruses- The DNA of certain viruses is also suitable for use as a vehicle DNA.

Bacteriophage DNA also used as a vector DNA for gene transfer.

Lambda phage (λ phage) has been used for transferring lac genes of E. coli into haploid callus of tomato.

Retro virus - useful for gene transfer in animal cell.

3. Desired DNA/ Alien DNA/ Foreign DNA/ Passenger DNA:-

It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. The passenger DNA can be complementary, synthetic or random.

i. Complementary DNA (cDNA)- It is synthesized on mRNA template with the help of reverse transcriptase and necessary nucleotides. cDNA formed through reverse transcription is shorter than the actual or in vivo gene because of the absence of introns or non-coding regions.

ii. Synthetic DNA (sDNA)- It is synthesized with the help of DNA polymerase on DNA template.

Kornberg (1961) synthesized first synthetic DNA.

Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the gene coding for yeast alanine t-RNA, which contained only 77 base pairs. However, it did not function in the living system. In 1979, Khorana was able to synthesis a functional tyrosine t-RNA gene of E. coli with 207 nucleotide pairs. Since then a number of genes have been synthesized artificially.

4. Host organism :- 

This organism is used for DNA cloning. Host may be plant, animal, bacteria (E.col. fungi (Yeast).

PROCESSES OF RECOMBINANT DNA TECHNOLOGY:

1. Isolation of DNA: The DNAs which are to be used as passenger DNA and the vehicle DNA are extracted out of their cells by lysing  the cells with the suitable enzyme.

The DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacterial), cellulose (plant cells), chitinase (fungus). You know that genes are located on long molecules of DNA intertwined with proteins such as histones.

The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed ty appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension DNA that separates out can be removed by spooling method.

2. Fragmentation of DNA by restriction endonucleases : Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme. Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.

Both the passenger and vehicle DNAs are then, cleaved by using the same restriction endonuclease so that they have complementary sticky ends.

Note : The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

3. Amplification of gene of interest using PCR

Polymerase chain reaction technology (PCR-technology)

This technique was invented by Kary mullis (1983).

In 1993 Kary Mullis got nobel prize for PCR (for chemistry).

PCR is a method for amplifying a specific region of DNA molecule without the requirement for time consuming cloning procedures.

PCR reaction takes place in Eppendorf tube.

Using PCR-technique very low content of DNA available from samples of blood or semen or any other tissue or hair cell can be amplified many times and analysed. In this technique Taq-Polymerase is used. Taq polymerase enzyme is used in PCR which is a special type of DNA polymerase enzyme which is resistant to high temperature.

Taq Polymerase is isolated from Thermus aquaticus bacteria.

Some other examples of polymerase which are used in PCR are-

Pflu Polymerase - Isolated from Pyrococcus furiosus bacteria.

Vent Polymerase - Isolated from Thermococcus litoralis bacterium

Main steps in PCR :-

Denaturation (94°) :- In this step a double stranded DNA molecule is placed at 94°C. So double stranded DNA becomes single stranded & each single stranded DNA functions as a template.

Annealing/Cooling (54°) - In this step two primer DNA are attached at 3' end of single stranded DNA.

Extension (72°):- In this process Taq polymerase enzyme synthesize DNA strain over template.

PCR is automatic process because Taq. polymerase enzyme is heat resistant.

4. Ligation of the DNA fragment into a vector- The complementary sticky ends of the passenger and vehicle DNAs are joined with ligase enzyme. This gives rise to a recombinant DNA.

5. Transferring the recombinant DNA into the host (Gene transfer):-

Transfer of desired genes from one organism into another is an important aspect of genetic engineering.

Gene transfer is achieved by two kinds of transfer methods:

1. Indirect method through vectors or carriers and

2.  Director vector less transfer method.

Indirect method :-

Gene Transfer in bacterial cell : Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made 'competent' to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by Incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.

Note: The bacteria to be used as hosts should be without plasmids. The host cells are treated with calcium chloride or lysozyme.

Transformant E.coli with plasmid.

Non-transformant E.coli without plasmid.

Gene Transfer in plant cell 

1. A plant pathogenic bacterium-Agrobacterium tumefaciens produces crown galls or plant tumours in almost all dicotyledonous plants.

2. This bacterium infects all broad leaved agricultural crops such as tomato, soyabean, sunflower and cotton but not cereals.

3. Tumour formation is induced by it's plasmid which is therefore called Ti plasmid (TI - tumour inducing) Agrobacterium tumefaciens naturally transfers some part of Ti-Plasmid into host plant DNA without any human effort so it is called natural genetic engineer of plant.

In the transformation process two essential component in Ti-plasmid -

 (a) T-DNA - (Transferred DNA)

(b) Vir-region-(Virulence region)

Inside the host plant cell T-DNA is seperated from Ti-plasmid, and integrated into host plant DNA that causes crown gall tumour.

Vir-region contains genes which are essential for T-DNA transfer and integration to host plant DNA.

When we use Ti-plasmid as a vector, first we remove the tumour causing gene from T-DNA region. Then desired gene inserted inplace of it. Now, this plasmid is called disarmed plasmid.

Same as Ri plasmid of A.rhizogenes (causing hairy root disease) also used as vector for gene transfer to plant cell.

Gene transfer in animal

Retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.

Direct method

Foreign genes can also be transferred directly by the following methods:-

a. Electroporation- It creates transient (temporary pores) in the plasma membrane to facilitate entry of foreign DNA.

b. Chemical mediated genetic transformation- It involves certain chemicals such as polyethylene gycol (PEG), that help in the uptake of foreign DNA into host cells.

c. Microinjection- it in the introduction of foreign genes mainly into animal cells using micropipette ar glass needles.

d. Particle gun/Biolistic method- It is a technique in which tungsten or gold particles coated with foreign DNA bombarded into target cells to facilitate entry of the foreign genes.

e. Liposome mediated gene transfer- In this method DNA encloses within lipid begs. Thest lipid begs funed with protoplast.

Selection of Transformed with recombinant cell:-

1. Selection by two antibiotic resistance gene 

You can ligate a foreign DNA at the Bam HI site of tetracycline resistance gene in the vector pBR322.

The recombinant plasmids will loss tetracycline resistance due to insertion of foreign DNA (insertional inactivation) now, it can be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants (plasmid transferd) growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non recombinants wil grow on the medium containing both the antibiotics. In This case, one antibiotic resistance gene helps in selecting the transformants.

Note:- 

Insertional inactivation- Due to Insertion of desired gene within selectable marker gene of vector, selectable marker gene become inactive or loose their Function. This is called Insertional inactivation.

2. Selection by one Lac Z gene and one antibiotics resistant gene-   Selection of recombinants due to inactivation of antibiotics is a cumbersome (troublesome) procedure because it requires simultaneous plating on two plated having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate X-gal gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase (reporter enzyme) and the colonies do not produce any colour, these are identified as recombinant colonies.

Obtaining the Foreign Gene Product :

When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial plant or animal cell the alien DNA gets multiplied. In almost all recombinant technologies, the ultimate aim is to produce a desirable protein. Hence, there is a need for the recombinant DNA to be expressed. The foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involve understanding many technical details.

After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale. Can you think of any reason why there is a need for large-scale production? If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein. The cells harbouring cloned genes of interest may be grown on a small scale in the 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 continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc. useing microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, Oxygen).

The most commonly used bioreactors are 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 through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

Downstream Processing -

After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished product.

The processes include separation and purification, which are collectively referred to as downstream processing.

The product has to be formulated with suitable preservatives. Such formulation has to undergo through clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing and quality control testing vary from product.to product.

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