Principles and Processes Chapter 11 BIOTECHNOLOGY Principles and Processes

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

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

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

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