1. Principles and Processes
Chapter 11
BIOTECHNOLOGY
Principles and Processes

2. BIOTECHNOLOGY
Deals with techniques of using live organisms to produce products and processes useful to humans.

3. EFB (European Federation of Biotechnology)
THE INTEGRATION OF NATURAL SCIENCE AND ORGANISMS, CELLS, PARTS THEREOF, AND MOLECULAR ANALOGUES FOR PRODUCTS AND SERVICES

4. PRINCIPLES OF BIOTECHNOLOGY
The two core techniques enabled birth of modern biotechnology
1. genetic engineering- 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 organisms.
2. chemical engineering- enable the growth of only desired microbe in large quantities.

5. PRINCIPLES OF BIOTECHNOLOGY
Sexual reproduction permits variation .
Asexual reproduction preserves the genetic information

6. PRINCIPLES OF BIOTECHNOLOGY
Traditional hybridisation very often leads to inclusion and multiplication of undesirable genes along with the desired genes.
Recombinant DNA , gene cloning and gene transfer- overcome this limitation and allows us to isolate and introduce only desirable genes.

7. PRINCIPLES AND PROCESSES
First instance of creating artificial recombinant DNA
1972- Stanley Cohen and
Herbert Boyer
Isolated DNA encoding for antibiotic resistance from Salmonella typhimurium
and transferred into E. Coli. bacterium

8. PRINCIPLES AND PROCESSES
Restriction enzymes-molecular scissors
Vectors
Ligases-molecular glues
Cloning- multiplication of copies of foreignDNA in host cell

9. PRINCIPLES AND PROCESSES
Three basic steps in genetically modifying an organism.
1.identification of DNA with desirable genes
2. introduction of the identified DNA into host
3. maintenance of introduced DNA in the host and transfer of the DNA to its progeny

10. PRINCIPLES AND PROCESSES
ORIGIN OF REPLICATION
The foreign DNA should be a part of chromosome having origin of replication
overview

11. TOOLS OF RECOMBINANT DNA TECHNOLOGY
RESTRICTION ENZYMES
POLYMERASE ENZYMES
LIGASES
VECTORS
HOST ORGANISMS

12. TOOLS OF RECOMBINANT DNA TECHNOLOGY
RESTRICTION ENZYMES
Are enzymes responsible for restricting the growth of bacteriophage (1963)
Restriction endonuclease

13. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Hind II- the first restriction endonuclease
They work at specific sequences of bases known as recognition sequences
There are over 900 restriction enzymes

14. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Naming a restriction enzyme
EcoRI-
First letter- of genera- eg: Escherichia
Second and third letters- of species- coli
Fourth letter- strain
Fifth roman number – the order in which the enzyme isolated

15. TOOLS OF RECOMBINANT DNA TECHNOLOGY
NUCLEASES
The class of restriction enzymes
Exonucleases- remove nucleotides
Endonucleases- cut DNA at specific sites

16. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Each restriction endonuclease works at palindromic sequences and make sticky ends -
MALAYALAM

17. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Working of restriction enzyme
cut little away from the centre of palindromic sequences leaving sticky ends
Stickiness is due to hydrogen bonds facilitates action of DNA ligaze
If same restriction enzyme is not used to cut vector and source DNA the recombinant molecule cannot be created

18. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Separation and isolation of DNA fragments
Cut DNA fragments can be separated by gel electrophoresis
DNA fragments are negatively charged.
Separated using a medium – usually agarose
Separated fragments visualised only after staining the DNA with a compound known as ethidium bromide and exposure to UV

19. TOOLS OF RECOMBINANT DNA TECHNOLOGY
ELUTION;
the separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.

20. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloning vectors
Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomes DNA.
-if an alien piece of DNA is linked with plasmid of bacteriophage we can multiply its numbers Equal to the copy number of the plasmid or bacteriophage.

21. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloning vectors
Features of vectors
1. origin of replication- sequence where replication starts
-- the selected origin or replication should support high copy number- since it controls the copy number

22. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloning vectors
2. selectable marker
Help in identifying and eliminating non-transformants and permitting the growth of transformants.
transformation
Are genes encoding resistance to antibiotics such as ampicilin, chloramphenicol etc.

23. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloning vectors
3. cloning sites
Cloning sites are recognition sites of restriction enzymes
At antibiotic resistant gene
pBR322

24. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloning vectors
3. cloning sites
Insertional inactivation
α- galactosidase

25. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Cloning vectors
3. vectors for cloning genes in plants and animals
Gene of interest can be introduced into plants or eukaryotic cell through vectors like pathogenic bacteria, virus like Agrobacterioum tumifaciens

26. TOOLS OF RECOMBINANT DNA TECHNOLOGY
Competent host
In order to force bacteria to take up the plasmid, the bacterial cells must first be made competent to take up DNA.
Microinjection
Biolistics or gene gun

27. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
ISOLATION OF DNA
FRAGMENTATION OF DNA
ISOLATION OF DERSIRED DNA FRAGMENT
LIGATION OF DNA INTO A VECTOR
TRANSFERING THE RECOMBINANT DNA INTO THE HOST
CULTURING OF HOST CELLS FOR LARGE SCALE EXTRACTION OF PRODUCT

28. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
1. ISOLATION OF THE GENETIC MATERIAL DNA
DNA SHOULD BE FREE FROM OTHER MACROMOLECULES
Treatment with lysozyme (bacteria),
cellulase ( plant cells)
chitinase (fungus)
RNA removed by ribonuclease and proteins by protease
DNA precipitates after addition of chilled ethanol
spooling

29. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Cutting of DNA at specific locations
Incubating purified DNA with restriction enzymes
The joining of DNA is done by mixing gene of interest, cut vector and ligase enzyme

30. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
3. amplification of GENE OF INTEREST using PCR
WITH primers and DNA polymerase enzyme.
The enzyme is thermostable from Thermus aquaticus

31. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Insertion of recombinant DNA into the host cell/organism
The resistance to antibiotics will act as selectable marker

32. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Obtaining the foreign gene product
Recombinant protein- heterologous host

33. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
BIOREACTORS\
Are used for large scale culture of host cell

34. PROCESSES OF RECOMBINANT DNA TECHNOLOGY
DOWN STREAM PROCESS
Purification of the biological product