Principles and Processes Chapter 11. BIOTECHNOLOGY Deals with techniques of using live organisms to...

BIOTECHNOLOGY

Principles and Processes

Chapter 11

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.


Sexual reproduction permits variation

.Asexual reproduction preserves the genetic information


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 PROCESSESRestriction enzymes-molecular sc

issors

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

The foreign DNA should be a part of chromosome having origin of replication

ORIGIN OF REPLICATION

overview

TOOLS OF RECOMBINANT DNA TECHNOLOGY

LIGASES

RESTRICTION ENZYMES

POLYMERASE ENZYMES

VECTORS

HOST ORGANISMS


RESTRICTION ENZYMES

Are enzymes responsible for restricting the growth of bacteriophage (1963)

Restriction endonuclease


Hind II- the first restriction endonuclease

•They work at specific sequences of bases known as recognition sequences

There are over 900 restriction enzymes


Naming a restriction enzyme

EcoRI-First letter- of genera- eg: Escherichia

Second and third letters- of species- coliFourth letter- strain

Fifth roman number – the order in which the enzyme isolated


NUCLEASES The class of restriction enzymes

Exonucleases- remove nucleotides

Endonucleases- cut DNA at specific sites


Each restriction endonuclease works at palindromic sequences and make sticky ends -

MALAYALAM


Working of restriction enzyme

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

cut little away from the centre of palindromic sequences leaving sticky ends


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


ELUTION;

the separated bands of DNA are cut out from the agarose gel and extracted from the gel piece.


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.


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


Cloning vectors

2. selectable markerHelp in identifying and eliminating non-transformants and permitting the growth of transformants.

transformation

Are genes encoding resistance to antibiotics such as ampicilin, chloramphenicol etc.


Cloning vectors

3. cloning sitesCloning sites are recognition sites of restriction enzymes

At antibiotic resistant genepBR322


• Cloning vectors

• 3. cloning sites

• Insertional inactivation• α- galactosidase


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


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

ISOLATION OF DNA

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

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

1. ISOLATION OF THE GENETIC MATERIAL DNADNA SHOULD BE FREE FROM OTHER MACROMOLECULES


DNA precipitates after addition of chilled ethanol

Treatment with lysozyme (bacteria), cellulase ( plant cells) chitinase (fungus)

RNA removed by ribonuclease and proteins by protease

spooling

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

3. amplification of GENE OF INTEREST using PCR


WITH primers and DNA polymerase enzyme.

The enzyme is thermostable from Thermus aquaticus

Insertion of recombinant DNA into the host cell/organism


The resistance to antibiotics will act as selectable marker

Obtaining the foreign gene product


Recombinant protein- heterologous host

BIOREACTORS\ Are used for large scale culture of host cell


DOWN STREAM PROCESS Purification of the biological product


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