Notes

BIOTECHNOLOGY: PRINCIPLES AND PROCESSES
Extra info: (The term BIOTECHNOLOGY was given by Karl Ereky in 1919)
(Paul berg was the first to transfer gene of SV-40 virus into E. coli through Lambda Bacteriophage. He received a Nobel prize for this in 1980)
Herbert Boyer found about restriction endonuclease from e coli while Stanley Cohen found how to extract plasmid one cell and transfer it to another, they combined their knowledge and made basis of biotechnology.

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

In this sense making curd (lactobacillus) bread or wine (cerevisiae sp.) which are all microbe-mediated processes could also be thought as a form of Biotechnology.

Other examples of biotechnology are in-vitro fertilization leading to "test tube baby", correcting a defective gene, developing a DNA vaccine and synthesizing a gene and using it.


The European Federation of Biotechnology has given a definition of biotechnology that encompasses both the traditional views and the modern 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"

PRINCIPLES:
Two core techniques that enabled the birth of modern biotechnology:
Genetic Engineering: techniques to alter the chemistry of the genetic material (DNA, RNA) and to introduce these into the host organism thereby changing the phenotype of the host organism.

Maintaining a sterile (microbial contamination free) ambience to grow only the desirable microbes/eukaryotic cell in large quantities to produce biotechnological products like antibiotics, vaccines etc.

Sexual reproduction has an advantage over asexual because it provides an opportunity for variation and formulates unique combinations of genetic set up however asexual preserves genetic information.

The traditional methods of hybridization of breeding of plants and animals often led to the inclusion and multiplication of undesirable genes along with desirable genes. However, the modern biotechnology for the creation of recombinant DNA, by using gene cloning and gene transfer overcomes this limitation and allows us to isolate and introduce only the desirable genes.

A piece of DNA can only multiply in a host organism if it is integrated into the genome of the host organism Ie. becomes a part of the chromosome. The chromosome has a specific sequence of DNA called the Origin of Replication which is responsible to initiate the process of replication.
Alien DNA has to be linked with ORI so that it can multiply and make copies of itself in the host organism. This can also be called as gene cloning or making multiple identical copies of any template of DNA.

The making of the first recombinant DNA emerged due to the possibility of linking a gene coding for antibiotic resistance with the native plasmid of Salmonella Typhimurium. This was accomplished by Stanley Cohen and Herbert Boyer in 1972. They were able to accomplish this by cutting a piece of DNA from the plasmid that was resistant to antibiotics.

Cutting of DNA at specific positions was possible due to the discovery of the so called "molecular scissors" Ie, Restriction Endonuclease. The cut DNA is then linked to the plasmid DNA. This plasmid DNA acts as a vector to transfer the desired gene to the host organism.

The linking of the DNA piece to the plasmid is with the help of linking enzyme DNA ligase also knows as the "molecular glue", this acts on the DNA molecules and joins the ends. This makes the combination of a new circular autonomously replicating plasmid.

When this DNA is transferred in E. coli it can replicate using E. coli's DNA polymerases and make multiple copies of itself. This ability to multiply to form multiple copies of the antibiotic resistant gene is called cloning of antibiotic resistant gene in E. coli.

Three basic steps in genetically modifying an organism:
1) identification of desirable genes
2) introduction of identified DNA into the host organism
3) maintenance of the introduced DNA in the host organism and transfer to progeny.

Restriction enzymes cut the two strands of the DNA a little away from the center but between the same bases, this leaves single strands at the ends of the DNA. These are overhanging stretches or sticky ends. They are called so because they form hydrogen bonds with their complimentary cut counter parts. The stickiness of these ends facilitates the action of the enzyme DNA ligase and helps to form the recombinant DNA.

When cut by the same restriction enzyme the two strands can be connected because they have the same kind of sticky ends.

The cutting of DNA leads to the formation of DNA fragments, these fragments of DNA can be separated by using a technique called Gel Electrophoresis. DNA fragments being negatively charged can be forced to move towards the anode under the influence of an electric field through a medium/matrix. The lightest molecules move the farthest distance. The most popular matrix/medium being used is the Agarose gel, this gel is a natural polymer which is extracted from sea weeds.

The separated DNA fragments can only be seen after staining the fragments with Ethidium Bromide followed by exposure to UV radiation. These fragments are then cut out from the agarose gel and then extracted from the gel piece this step is known as Elution. The DNA fragments purified in this way can be used the construction of the recombinant DNA.

Plasmids and bacteriophages have the ability to replicate in the cell independent of the control of the chromosomal DNA. The bacteriophages being high in number have a high copy number. Some may have one to two copies per cell while some might have 15-100 copies, many might have even higher.

Featured that are required for cloning:
1) Origin of replication: This is a sequence of DNA present on the chromosomes where the process of replication is initiated. Any fragment of DNA can be made to multiply within the host organism when linked to the ori site of the organism. This site is also responsible for controlling copy number of the plasmid hence if a larger copy number is needed then an ori with a high copy number must be selected.
2) Selectable Markers: These are used to select transformants from non-transformants. An example is genes that show resistance to antibiotics like ampicillin, kanamycin, chloramphenicol, tetracycline etc. are considered useful selectable markers for E. coli cell. A normal E. coli cell does not carry resistance against any of these antibiotics.

3) Cloning sites: In order to link the alien DNA, the vector needs to have recognition sites (preferably few). A vector must have recognition sites for many enzymes but only 1 for any particular enzyme otherwise the DNA will divide into multiple fragments, and it will complicate gene cloning.
The foreign DNA is ligated at the recognition site of the vector. Recombinants and transformants can be selected with the help of selectable markers. Take an example for the vector pbr322 which is an artificial vector plasmid. An alien gene is ligated at the BAM H1 site of the plasmid. This site carried resistance to tetracycline, now since another gene is placed here the new plasmid loses resistance for tetracycline. The recombinants can be selected from non-recombinants by placing the transformants on a plate containing ampicillin medium. The recombinants will show resistance to ampicillin while the non-recombinants will not. These are then transferred to a plate contaning tetracycline medium, the recombinants will not grow in the medium containing tetracycline while non-recombinants will grow in both the mediums. From this we can see that one antibiotic resistance gene helps to select transformants from non-transformants while the antibiotic resistance gene gets inactivated due to the insertion of alien DNA and helps in selection of recombinants from non-recombinants.

Selection of recombinants because of inactivation of antibiotics is a cumbersome process because it requires simultaneous plating's containing different mediums of antibiotics hence alternative selectable markers have been developed which help in selecting recombinants from non-recombinants based on their ability to produce color in the presence of a chromogenic substrate. In this a recombinant DNA is inserted into the coding sequence of an enzyme B-Galactosidase this results in inactivation of the enzyme and is called "Insertional Inactivation". The presence of this enzyme would have given blue colored colonies in the presence of a chromogenic substrate. However, after inserting the recombinant DNA this enzyme gets inactivated and gives colorless colonies. The presence of colored colonies helps in selecting recombinants from non-recombinants.

Agrobacterium Tumefaciens, a pathogen to many dicot plants has the ability to transfer a piece of DNA called "T-DNA" into the plant cells and transform normal plant cells into tumor and direct them to produce chemicals required by the pathogen.

Retrovirus also have a similar ability. They can transform normal animal cells into cancerous cells.