Biotechnology : Principles and Processes

1. Biotechnology : Principles and Processes Prepared by: Mrs. PriyankaTyagi PGT (Biology) K.V. Dogra Lines Meerut Cantt

2. What is biotechnology? • Biotechnology refers to the technology using biology, which has applications in agriculture, food processing industry, medicine diagnostics, bioremediation, waste treatment, and energy production.

3. Biotechnology • Biotechnology deals with  the techniques of using live organisms or enzymes from organisms to make products and processes that benefit human beings. • The definition given by European Federation of Biotechnology (EFB) is as follows: “The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.”

4. Principles of Biotechnology • Genetic engineering : Introduction of foreign genetic material (DNA/RNA) into the host’s genome and altering its phenotype. • Aseptic techniques : Maintenance of sterile atmosphere to enable growth of only the desired cell in large quantities for the manufacture of products like antibiotics, vaccines enzymes etc.

5. Techniques Of Genetic Engineering • Creation of recombination DNA • Gene transfer into host organism • Gene cloning

6. Creation Of Recombination DNA • Stanley Cohen and Herbert Boyer (1972) constructed the first recombination DNA. • They isolated the antibiotic resistance gene from the Plasmid of the bacterium Salmonella typhimurium. • This piece of DNA carrying antibiotic resistance gene was cut at specific location by restriction endonuclease, popularly known as Molecular Scissors.

7. Gene transfer • The cut piece of DNA was introduced in the plasmid of Escherichia coli which acted as the vector. • The piece of DNA was ligated to the vector plasmid by DNA ligase. • This joining of the two DNA pieces resulted in the creation of recombinant DNA.

8. Gene cloning • The new recombinant DNA was transferred into E. coli. • The r DNA replicated autonomously by using the host DNA polymerase enzyme and made multiple copies. • The ability to multiply copies of any template of DNA is called gene cloning.

9. Tools of Recombinant DNA Technology • Restriction enzymes • Cloning Vector • DNA polymerase enzyme • DNA Ligase enzyme • Host organism

10. Restriction enzymes As biological scissors !!!!

11. Restriction enzymes • Restriction enzmes belong to a class of enzymes called nucleases. • These are of two types – exonucleases and endonucleases. • Exonucleases cut the DNA at the ends. • Endonucleases make cuts at specific points in the DNA.

12. Restriction enzymes • The first restriction endonuclease isolated – Hind II. • It was isolated from the bacterium Haemophilus influenzae. • Today we know more than 900 restriction enzymes isolated from 230 strains of bacteria.

13. Recognition sequence • Restriction enzymes always cut DNA molecules at a particular point by recognizing a specific sequence of base pairs. • This specific base sequence is known as recognition sequence. • Example: EcoRIrecognises only the following sequence. 5’-------GAATTC-------3’ 3’-------CTTAAG-------5’

14. Restriction Enzymes…….How do you denote them???? • Names use 3-letter italicized code: • 1st letter - genus • 2nd & 3rd - species • Following letter denotes strain EcoRI was the first restriction enzyme found in the R strain of E. coli

15. PalindromicSequence • Palindromes are those group of letters which read the same, both forward and backward. Ex. MALYALAM • A Palindromic sequence is a sequence which reads the same on the two strands of DNA when orientation is kept the same. 5’-------GAATTC-------3’ 3’-------CTTAAG-------5’

16. Cutting & Pasting…….Restriction enzymes as molecular scissors • Restriction enzymes • Three types: Types I, II and III • Type IIused for molecular biology work • Endonucleases • Recognize specificsequence of base-pairs, usually 4, 6 or 8 bases that are palindromic • Can leave ‘sticky ends’ or ‘blunt ends’

17. 5’ ATGCGAATTCCGGAA 3’ 3’ TACGCTTAAGGCCTT 5’ 5’ ATGCGATATCCGGAA 3’ 3’ TACGCTATAGGCCTT 5’ About sticky and blunt ends……… Sticky ends EcoR1 5’-AATTCCGGAA-3’ 3’-GGCCTT-5’ 5’-ATGCG-3’ 3’-TACGCTTAA-5’ Blunt ends EcoRV 5’-ATGCGAT-3’ 3’-TACGCTA-5’ 5’-ATCCGGAA-3’ 3’-TAGGCCTT-5’

21. What do restriction enzymes help us achieve ??? • They help in generating DNA fragments with precise ends • These precisely generated ends can then be ‘pasted’ to similar ends in a vector molecule that is self replicating. • The process of ‘pasting’ is called ligation and requires a DNA ligase • The product after ligation is called a ‘recombinant DNA molecule’ or a ‘clone’ • Each clone can be replicated to provide ample material for study

22. What Are Cloning Vectors? • Cloning vector is a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA. • Features of cloning vectors • Types of Cloning Vectors • General steps of cloning any vector

23. Features of Cloning Vectors • Origin of replication: a sequence from where replication starts. • Selectable marker: a method of selecting for bacteria containing a vector with foreign DNA; permits the growth of transformants and eliminate the non-transformants. usually accomplished by genes encoding resistant to antibiotics such as ampicillin, chloramphenicol,tetracycline or kanamycin. • Cloning site: to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes

24. It shows a typical plasmid vector.  It contains a polylinker which can recognize several different restriction enzymes, an ampicillin-resistance gene (ampr) for selective amplification, and a replication origin (ORI) for proliferation in the host cell.  

25. Plasmids or Bacteriophages are used as vectors. • If we are able the link an alien DNA with Bacteriophage or Plasmid DNA, we can multiply its number equal to the copy number of the Plasmid or Bacteriophage.

26. Vectors • DNA molecules capable of accepting a ‘foreign/new’ DNA fragment • Most common are plasmids but could be of other types also. • They are self replicating because they have an origin of replication (ori) • Plasmids are circular, double-stranded, extra-chromosomal pieces of DNA • Plasmids carry antibiotic resistance genes and a region to clone the ‘new’ DNA called the multiple cloning site

27. PLASMID • A plasmid is an independent, circular, self-replicating DNA molecule that carries only a few genes.

30. Vectors for cloning genes in plants • Agrobacteriumtumefacienscan cause the plant disease crown gall by transferring specific genes to the dicot plant. • A. tumefaciens contains a large plasmid called Ti plasmid which can deliver T- DNA to transform normal cell into a tumor and direct these tumor cells to produce the desired chemicals. • Plant genetic engineers have used this natural transformation system as a vehicle for the introduction of foreign DNA into plants.

31. So what is Agrobacterium????? A natural genetic engineer!! Agrobacterium tumefaciens is a soil bacteria that causes common plant tumours, commonly known as Crown Gall disease which affects a wide variety of plants. The genome ofAgrobacterium tumefaciens C58 has been sequenced completely and consists of a circular chromosome, a linear chromosome and two plasmids

32. A. tumefaciens gall is not a tiny thing!!!!!

33. T-DNA region Tumor- producing genes Opine catabolism Virulence region ORI The Ti Plasmid

34. During infection, the Ti plasmid is integrated into the plant chromosomal DNA

38. Produce callus  transform callus  stimulate shooting by cytokinin addition + cytokinin This procedure is easy for dicotyledon plants (legumes etc) Biology of Plants, Raven et. al., Freeman Worth Publishing, 1999

39. Monocotyledons are not easy to handle – callus is very difficult to be initiated, and A.tumefaciens is not pathogenic for them 1. Pericarp sholud be pulled back and the immature embryos (0.5 - 1.0 mm) are removed. 2. The immature embryos are placed on a callus induction medium Transformation is performed by gene gun method high osmotic media prepare calli for transfomation

40. After shooting calli are placed on a selective media containing a herbicide for three weeks. Then calli are transferred to a media to induce the production of shoots. After they form small shoots, they are transferred to DARKER containers on a root induction media.

41. Vectors for cloning genes in Animals • Retroviruses in animals have the ability to transform the normal cells into cancerous cells. • Retroviruses are disarmed and now used to deliver desirable genes into animal cells.

42. Competent Host(for transformation with r-DNA) • Chemical treatment: Bacteria cell is treated by calcium to make them competent. • Incubate the cells with r-DNA on ice, • heat shock by placing them at 42oC, • putting them back on ice. This enables the bacteria to take up the r-DNA.

43. Other methods • Micro injection: r-DNA is directly injected into the nucleus of an animal cell. • Gene gun or Biolistics: plant cells are bombarded with high velocity micro-particles of gold or tungstun coated with r-DNA. • Disarmed Pathogen: agrobacterium tumifaciens in plants and Retroviruses in animals.

44. Micro-injection: The host cell is immobilized by applying a mild suction with a blunt pipette. The foreign gene is then injected with a micro-injection needle.

45. Cell’s DNA DNA coated golden particles Gene gun Plant cell A plant cell with the new gene Cell division Transgenic plant “Gene Gun” Technique

46. DNA with desired gene and antibiotic resistance is coated onto the surface of gold particles. Calli are placed in vacuum chamber, Helium pressure shot DNA into cells Genegun Coating gold particles with DNA vacuum chamber Calli remain on the high osmotic media for 20 hours following shooting.

47. Closer look on: “gene gun”

48. Processes of Recombinant DNA Technology • Isoaltion of the Genetic Material(DNA) • Cutting of DNA at specific location • Amplication of Gene of Interest using PCR • Insertion of r-DNA into the Host cell/organism • Obtaining the Foreign Gene Product

49. Isoaltion of the Genetic Material(DNA)