GENETIC ENGINEERING

BY: RAHUL ANDHARIA (MSIWM001)

Genetic Engineering: It refers to altering the genetic material of an organism by insertion or removal of individual genes from another organism. In other terms it simply means manipulation of genetic make-up of an individual by artificial means. The organism that receives the genetic material is termed as transgenic organism.

History:

Father of Genetic engineering is Paul Berg. The process of Genetic engineering was first showcased by Paul Berg when he introduced the viral gene in bacteria with the help of lambda phage. Transgenesis, process of transferring of genes from one organism to other was first pioneered by Herbert Boyer and Stanley Cohen, in the year 1973.

Principle of Genetic Engineering:

There are several steps associated with Genetic Engineering:

1. Isolation of gene of interest to be cloned from desired organism.
2. Transfer of the isolated gene to create recombinant DNA molecule. This can be done by cutting the DNA molecules at specific sites by using Restriction Enzymes.
3. Restriction enzymes cut DNA at specific locations. There are basically 4 types of restriction enzymes designated as I, II, III, and IV. All this four enzymes primarily differs in their structure, site of cleavage and some cofactors (substance which is required for enzyme activity).
4. By the action of RE, DNA is left with sticky ends (short portion of unpaired bases). A bacterial plasmid is also cut with the same RE, so that the plasmid also has the similar sticky ends and can base pair. (Plasmids are extra-chromosomal DNA found inside the bacterial cell).
5. The plasmid and the isolated gene are joined together by an enzyme called DNA ligase.
6. The two pieces of DNA with same sticky ends (as cut by same RE) are being linked together by DNA ligase and forms a single, unbroken molecule.
7. Than the genetically engineered plasmid is inserted into bacterial cell (Transformation). The gene insertion usually takes place by means of Vectors. (Molecules that carry the gene of interest.) choice of vectors depend on the type of gene to be inserted.
8. When the bacteria reproduce, the plasmid will get copied and this recombinant plasmid spreads as bacteria multiply and expresses the gene and makes a human protein.

This is the basic procedure required to obtain a desirable gene and this whole process together refers to Genetic Engineering.

Various types of Genetic engineering techniques:

1. Recombinant DNA technology:

Technique in which artificial DNA fragment is created by ligation of two different DNAs using various physical methods.

In this technique basically the gene of interest is inserted into a plasmid vector and can be used for gene transfer experiments. Two methods are commonly in use which includes:

1. Plasmid method- In this method small pieces of circular DNA called plasmids are used for altering microorganisms like bacteria.
2. Vector method- vectors are small carrier molecules that carry viruses. Generally, viruses have capsule, in which the DNA is present. When the viruses attach to the host cell they insert its DNA or RNA. The DNA in the host genome replicates itself by using host machinery and the gene inserted will become part of the host machinery.

• Gene delivering:

This technique is employed to insert gene of interest directly into host genome. Some of the most popular gene delivering techniques which include:

1. Electroporation- introduction of DNA into cells by using electric current. It is a rapid method to create more number of recombinants within a shorter duration of time.

The mixture containing cells and DNA are placed in a glass chamber. The glass chamber contains two electrodes. A single electric pulse of about 4000-8000 volts/cm is generated between the two electrodes for about 4-5 milliseconds. The electric current forms transient pores in the cell membrane through which the DNA seems to enter into the cells.

• Liposome mediated gene transfer: liposome is a small vesicle made up of phospholipids used to deliver foreign DNA into cells. Lipids like phosphetidyl choline, Cholesterol are useful in making liposomes. Outer lipid bi-layer generally fuses with the cell membrane and releases the contents.

• Gene editing:

The technique is used to edit the genome in which an undesired or diseased gene can be edited or replaced by a new gene in the host genome.

Some of the gene editing systems used are: CRISPR-CAS9, TALEN( Transcriptional activator like effector nuclease) and  ZFN( zinc finger nuclease).

Among this three CRISPR is the most widely used method currently. Bacteria generally possess this method in their genome to combat viruses. CRISPR normally  stands for clustered regulatory interspaced short Palindromic repeats. The nuclease, CAS9 cuts the DNA strands of foreign DNA and pathogens and thus destroys them.

Process overview of Genetic engineering:

• Isolation of desired gene
• Selection and plasmid construction
• Gene transformation(uptake of DNA by cells)
• Insertion of DNA into a suitable host
• Insert confirmation- done by using various selectable markers.

Applications and future aspects of genetic engineering:

Genetic engineering has great prospects. It is highly practiced in Industrial and agricultural field. It has wide range of applications in the field of medicine, genetic research, and in therapeutic drug production.

1. Plant Genetics: It is highly employed in plants to improve crops. Recombinant DNA technology in plants help to have better yield, to control pathogens, delayed fruit ripening, stress tolerant plants.

Classic example is Bt cotton, most famous Genetically modified crop.

• Genetically engineered Insulin: human insulin gene is inserted into the gap in plasmid. The plasmid becomes genetically modified. The plasmid than upon insertion into new bacteria or yeast cell will produce insulin.