Biotechnology -principles And Process

BIOTECHNOLOGY : PRINCIPLES AND PROCESSES Biotechnology is the techniques of using live organisms or their parts to produce products and processes useful to humans EFB (European Federation of Biotechnology) definition of biotechnology The integration of natural science and organisms,cells, parts thereof, and molecular analogues for products and services. Important terms in G.E. Recombinant DNA(rDNA)-DNA made by join the fragments of DNA from different organisms. Gene cloning-The process of making multiple identical copies of gene Gene transfer-The process of transfer of a desired gene from one organism to another Plasmid- Self-replicating circular extra-chromosomal DNA Cloning vectors-carriers of a desired gene which are used to transfer from one organism to another

PRINCIPLES OF BIOTECHNOLOGY (i) Genetic engineering : Genetically modifying an organism (ii) Maintenance of sterile condition in all processes G.E also called Recombinant DNA Technologyy(rDNA technology) because it produce recombinant DNA.Stanley Cohen &Herbert Boyer first rDNA produced in1972 by inserting the antibiotic resistance gene in to the plasmid of Salmonella typhimurium. Basic techniques of G.E. 1) Creation of Recombinant DNA 2)Gene cloning 3)Gene transfer Importance G.E. G.E. help us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into organism. TOOLS OF RECOMBINANT DNA TECHNOLOGY/G.E a) Restriction enzymes b) Polymerase enzymes c) Ligases d) Cloning vectors e) Host organism. a)Restriction Enzymes (molecular scissors, molecular knife ) Enzyme which cut DNA at specific points called restriction enzymes. Restriction enzymes are nuclease enzymes. These are of two kinds - Exonucleases - make cut at the end of the DNA. Endonucleases- make cuts within the DNA. Common REs are restriction endonucleases. The first restriction endonuclease is Hind II Recognition sequence- The specific DNA sequence required for identification of cutting site for REN.This sequence is a palindromic nucleotide sequence.Palindromes that form the same words when read both forward and backward. e.g., "MALAYALAM". 5` -- GAATTC -- 3` 3` -- CTTAAG -- 5 Naming of restriction enzymes -The first letter from the genus name -The second two letters from the species name, - Fourth letter from the strain name . - Last Roman number denote order number of REs, from which they were isolated Eg.Escherichia coli RY 13. ie. EcoRI Action of REs a) Identification of recognition sequence in the foreign DNA and vector DNA molecule b) Make a cut and producing sticky end at both sides of foreign DNA and vector DNA by using same RE. (Figure 11.1) Separation and isolation of DNA fragments -The fragmentes of DNA can be separated by gel electrophoresis. -Commonly used gel is agarose gel . -DNA frangments are seperated based on their charges and sizes. -The separated DNA fragments stained with ethidium bromide are exposed to UV light produces a bright orange coloured DNA bands . -The isolation of desirable DNA fragment from the gel is known as elution. b)Ligases (molecular glue) Enzyme joined two molecule Eg. DNA ligases c)Polymerases Enzymes synthesis new molecules Eg. DNA polymerases d) Cloning Vectors Cloning Vectors DNA fragment used as a carrier for transfering desired gene into the host organism.The main functions of CV are , -transfering gene of interest into host cell -storing gene of interest -multiplication of gene of interest Commonly used cloning vcters are a)Plasmid vector; . Eg. pBR322 b)Phage vector;DNA of bacteriophage . Eg.lamda phage DNA Features of a cloning vectors i)Origin of replication(Ori) ii)Selectable Marker iii) Cloning sites (i) Origin of replication (ori) The DNA sequence responsible for starting of replication is called Origin of replication. It help to produce many copies of the desired DNA . (ii) Selectable marker Selectable markers are gene which help identification and elimination of nonrecombinants from recombinants. The genes encoding antibiotic resistance and colour reaction used as Selectable markers (iii) Cloning sites The recognition sites for the restriction enzymes in cloning vector ,are called Cloning sites. The cloning site present in one of the selectable marker genes is good for identification of recombinants Presence of more than one recognition sites for a restriction enzyme in a vector cause DNA fragmentation and gene cloning become difficults.So,one site for one RE. Selection of Recombinants 1.Steps for Antibiotic resistance selection a)Ligate a foreign DNA at the Bam H I site of tetracycline resistance gene in the vector pBR322 . b)Then grow the all bacteria cells in 2 culture medium (ampicillin containing medium& tetracycline containing medium) by replica plating method.We will get following results. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, nonrecombinants will grow on the medium containing both the antibiotics. Based on above results, we can select the recombinant & non recombinant bacterial cells.

2.Steps for colour reaction selection. Alternative selectable markers have been developed on the basis of their ability to produce colour in the presence of a chromogenic substrate. Steps # A foreign DNA is inserted within the gene sequence of an enzyme, -galactosidase of plasmid vector. Inactivation of the enzyme production in the bacterial cells due to the insertion is insertional inactivation # Then grow the baterial cells in the presence of a chromogenic substrate in the medium.Results are Nonrecombinants gives blue coloured colonies . Recombinants never produce any blue colour due to insertional inactivation of the gene of ?-galactosidase Gene transfer in plants and animals : There are various procedure for transferring genes into plants and animals from bacteria and viruses.The process of transfer of a desired gene from one organism to another is called gene transfer It maytake place in 2 ways . They are vector-through gene transfer & vectorless gene transfer . 1.Vector-through gene transfer The process of gene transfer through vectors is called vector-through gene transfer.It is indirect gene transfer. Agrobacterioum tumifaciens tumor inducing (Ti) plasmid is a commonly used plasmid vector in plants. Ti plasmid has an important transferable part called T-DNA .Non pathogenic Agrobacterioum Ti plasmid haveT-DNA used to deliver genes of our interest into a variety of plants.The gene transfer through Agrobacterioum is called Agrobacterioum mediated gene transfer.Steps involved in Agrobacterioum mediated gene transfer are -The desired gene is to be inserted withinT-DNA of Ti plasmid of disarmed Agrobacterioum -Ti plasmid with desired gene inserted into the plant cell by disarmed Agrobacterioum -The T-DNA along with desired gene is separated and integrated within the DNA of plant cell and produce desired gene product in them Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells.So ,they have also been disarmed and are now used to deliver desirable genes into animal cells. 2.Vectorless gene transfer The process of gene transferance directly into host cells is called vectorless gene transfer. It is direct gene transfer.Recently two new methods are developed- micro-injection and biolistics or gene gun a.Micro-injection:Insertion of foriegn DNA is directly injected into the nucleus of an animal cell using micropipettes is called micro-injection . b. Biolistics or gene gun: In this method, high velocity micro-particles of gold or tungsten coated with desired DNA are bombarded into the plant cells.The presence of cell wall in the plant cell this method is suitable for plants e) Competent Host The cell which is cabale of taking up foreign DNA is called Competent Host. DNA is a hydrophilic molecule, it cannot pass through cell membranes because of the membranes are hydrophobic in nature.So, the host cell made competent in order to take up the recombinant DNA.. This is done by treating them in following ways. -The host cell treated first with a divalent cation,such as calcium, which increases the entery of DNA through cell wall. -Recombinant DNA can enter through cell membranes by 1.Incubating the cells with recombinant DNA on ice. 2.It is followed by placing them briefly at 420C (heat shock), and then putting them back on ice. This enables the bacteria cell to take up the recombinant DNA.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY/ GENETIC ENGINEERING Recombinant DNA technology involves several steps in specific sequence such as 1 Isolation of the Genetic Material (DNA) In order to isolate a pure form DNA from a cell following steps are needed. a.Treated the cells with enzymes such as lysozyme(bacteria),cellulase(plant cells), chitinase (fungus) for breaking the cell wall/cell membranes of the cell b. Other macromolecules such as RNA, proteins,polysaccharides and lipids are removed by treating them with specific digestive enzymes like protease ,ribonuclease,lipase etc. c. This fine threads of DNA can be removed by spooling(it is a process by which DNA threads wind on a reel 2 Cutting of DNA by using R.Es Purified DNA molecules treated with the restriction enzyme, in the optimal conditions for making DNA fragments 3 Isolation of the desired DNA fragment The seperation of the DNA fragments and isolation of the desired DNA fragment by using Agarose gel electrophoresis . 4 Amplification of Gene of Interest using PCR The process of making many copies of a gene is called amplification of Gene.It is done by using Polymerase Chain Reaction or PCR. In this reaction, following components are required -two sets of primers (small oligonucleotides that are complementary to the regions of DNA) -the enzyme DNA polymerase - four types of nucleotides. PCR has 3 basic steps- (i)Denaturation;(ii) Primer annealing (iii) Extension of primers (i)Denaturation The seperation of 2 strands of fragment by treating with high temperature (90-95?C).Each single strand of the DNA acts as a template for DNA synthesis. (ii) Primer annealing Here,2 primers anneal to each of the single stranded DNA at its 3'end.Annealing occurs by low temperature treatment(40-60 ?C). (iii) Extension of primers DNA polymerase enzyme extends the primers using the four types of nucleotides.This process is called Extension of primers or Polymerisation.Commonly used DNA polymerase for PCR is thermostable (active in high temperature) Taq polymerase (isolated from a bacterium,Thermus aquaticus). This3 steps are repeated many times and DNA amplified in the 2? pattern. 5 Ligation of the DNA fragment into a vector Amplified fragment used to ligate with a vector by using DNA ligase and form recombinant DNA 6 Insertion of Recombinant DNA into the Host Cell/Organism Recombinant DNA bearing gene for selectable marker is transferred into competent a bacterial, plant or animal cell . 7 Obtaining the Foreign Gene Product in large scale Always a gene product in G.E. is a desirable protein useful to humans. The protein produced by the through recombinant DNA , is called a recombinant protein .Their large scale production by using bioreactors. The device in which large scale production by using living cells and their growth medium,is called bioreactors or fermenter. The most commonly used bioreacters are of stirring type, which are shown in Figure 11.7 Significance of bioreactors - Large -scale production of desired proteins under aseptic condition -It is provide many controlled monitoring systems -It providing optimum growth conditions 8 Downstream Processing The process of separation and purification of a biosynthetic product is called downstream processing.The important steps in the downstream processing are, 1. Separation of gene product 2. Purification of gene product 3. Addition of suitable preservatives for avoid decay 4. Clinical trials for medicinal purposes 5. Quality control testing