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principles and processes

What is Biotechnology? use of living organisms and their products to modify human health and the human environment.
Who gave the definition of biotechnology? The European Federation of Biotechnology (EFB)
EFB definition of biotechnology The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.
Two core techniques of Biotechnology Genetic Engineering Techniques to alter the chemistry of genetic material thus change the phenotype of the host organism. Microbial contamination-free ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell
Cloning Cloning is making multiple identical copies of any template DNA.
What made construction of first recombinant DNA possible? The construction of the first recombinant DNA emerged from the possibility of linking a gene-encoding antibiotic resistance with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of Salmonella typhimurium.
Who first isolated the antibiotic resistance gene by cutting out a piece of DNA from a plasmid? Stanley Cohen and Herbert Boyer in 1972
Vectors plasmid DNA act as vectors to transfer the piece of DNA attached to it into the host organism.
DNA ligase enzyme The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase which acts on cut DNA molecules and join their ends.
Three basic steps in genetically modifying an organism. 1) identification of DNA with desirable genes. 2) Introduction of the identified DNA into the host. 3) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
TOOLS OF RECOMBINANT DNA TECHNOLOGY 1) DNA manipulative enzymes (i) Restriction enzymes (ii)Polymerase enzymes (iii)Ligases enzymes 2) Vectors 3) Host organism
Restriction Enzymes (RE) In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in E. coil were isolated. One of these added methyl groups to DNA (methylase), while the other cut DNA. The later was called restriction endonuclease.
Who discovered RE? Werner Arber, Dan Nathans and Hamilton Smith
Main Uses of RE Restriction enzymes serve as chemical knives to cut genes (= DNA) into defined fragments.
Other uses of RE These may then be used to determine the order of genes on chromosomes or to analyze the chemical structure of genes and of regions of DNA which regulate the functions of gene or to create new combinations of genes.
First RE The first restriction endonuclease Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later. Restriction enzymes belong to a larger class of enzymes called Nucleases.
Define Recognition sequence of Hind II It was found that Hind II always cut DNA molecules at a particular point by recognizing a specific sequence of six base pairs.-This specific base sequence is known as the recognition sequence for Hind II.
Recognition sequence for Hind II 5' GT (Pyrimidine :T or C) (Purine : A or G) AC3' 3' CA (Purine A or G) (Pyrimidine :T or C) TG5'
Name convention for RE first letter comes from the name of the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated.
Meaning of name EcoRI EcoRi comes from Escherichia coli RY13. In EcoRl, the letter 'R' is derived from the name of strain "Rough". Roman number following the names indicate the order in which the enzymes were isolated from that strain of bacteria.
Exonucleases removes nucleotides from the end of a DNA molecule.
Endonucleases removes nucleotides by breaking internal phosphodiester bonds.
Palindrome palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept same. These sequences may range from 4-8 nucleotides in length.
Types of RE and the most used one. There are three types of restriction enzymes though most restriction enzymes employed in genetic engineering are Type II because they can be used in-vitro to recognise and cut within the specific DNA sequence typically consisting of 4—8 nucleotides.
Working of restriction enzymes RE functions by inspecting the length of a DNA sequence. once it finds its specific recognition sequence it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar phosphate backbone.
Sticky or cohesive ends formation RE of EcoRi cut the strand of DNA a little away from the centre of the palindrome sites but between the same two bases on the opposite strands this leaves single stranded overhanging structures called sticky ends.
Uses of sticky ends They form hydrogen bonds with their complimentary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.
Flush ends Some restriction enzymes cut the strand of DNA in the centre of palindrome such ends are called blunt ends or flush ends.
Function of DNA ligase This enzyme forms phosphodiester bonds between adjacent nucleotides and covalent links to individual fragments of double stranded DNA by utilising energy from cell the enzyme used most often in our DNA is T4 DNA ligase.
Function of DNA polymerase These enzymes synthesise a new strand of DNA complementary to an existing DNA template in 5' to 3' direction. Usually DNA polymerase I is employed in genetic engineering
Essential features of a vector Origin of replication selectable marker cloning sites size of vector
Origin of replication This is a sequence from where replication starts. An alien DNA is linked with the ori so that this alien piece of DNA can replicate and multiply itself in the host organism. It is also responsible for controlling the copy number of the linked DNA.
Selectable marker Helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. Normally the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol are considered useful selectable markers.
Selectable Marker for E coli Do not carry resistance against any of these antibiotics also the gene lac Z coding for beta galactosidase enzyme may also be used as a selection basis. This enzyme utilizes its substrate to produce a blue coloured product therefore it is chromogenic.
Cloning sites In order to link the alien DNA the vector needs to have very few perferably single recognition sites for the commonly used RE. Presence of more than one recognition site within the vector will generate several fragments which will complicate gene cloning.
Process of insertion of gene The gene of interest is inserted at RE site located in any one of the antibiotic resistance genes or selectable marker genes such that Recombinant vector will lose antibiotic resistance due to insertion of foreign DNA to one selectable marker.
Size of vector Should be small as large molecules have tendency to break down during purification
Examples of vectors commonly used in RDT Plasmids, bacteriophage, cosmid, YAC vector, BAC vector phagemid,transposons as vector, shuttle vectors.
Plasmids Extrachromosomal circular, nonessential, double standard, autonomous, self replicating pieces of DNA in bacterial and some yeast cells. May confer the property of antibiotic resistance in bacteria and virulence in Ti plasmid in agrobacterium tumefaciens.
Restriction sites of E coli cloning vector pBR322 Hind III, EcoR I, BamH I, Sal I, Pvu Ii, Pst I, Cla I
Antibiotic resistance genes of e coli amp^R and tet^R
What does rop codes for? Proteins involved in the replication of the plasmid
Explanation of the name pBR322 p- plasmid BR- Boliver and Rodriguez 322- distinguishes this plasmid from others developed in the same laboratory
Features of plasmid DNA Always double standard, circular, naked without histone protein, does not carry any vital gene necessary for cell, can replicate independent of main genome, introns are absent.
Features of chromosomal DNA Maybe single stranded or double standard, linear or circular, coated with histone protein, carries vital genes necessary for cells, replicates with genome, both exons and introns are present.
Characteristics of pBR322 Size = 4.3 KB two sets of antibiotic resistance gene therefore selection is a two-step process, reasonably high copy number
Selection using antibiotic resistance of pBR322 One antibiotic resistance gene helps in selecting the transformants, whereas The Other antibiotic resistance gene gets inactivated due to insertion of alien DNA and helps in selection of recombinants.
Disadvantage of pBR322 Selection of recombinants requires simultaneous plating on two plates having different antibiotics.
Advantage of pUC8 over pBR322 Higher copy number Identification of Recombinant cells achieved by a single step.
How does selectable marker in pUC8 differentiates recombinants from non recombinants? On the basis of their ability to produce colour in the presence of a chromogenic substrate. Presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert.
Bacteriophage Virus that infect bacteria and has the ability to replicate within bacterial cells independent of the control of chromosomal DNA
Copy number of bacteriophage Because of their high number per cell they have very high copy number of their genome. If we link and alien piece of DNA with bacteriophage we can multiply its numbers equal to the copy number of the bacteriophage
Most commonly used bacteriophages Lambda phage vector and m13 phage vector
Lambda phage vector It allows cloning of DNA fragments upto 23 KB length
M13 phage vector Filamentous phage which infects E coli. Foreign DNA can be inserted into it without disrupting any of essential genes
Cosmid Combination of Cos site of Lambda phage and plasmid DNA
YAC vector Yeast artificial chromosome contain telomeric sequence, the centromere and autonomously replicating sequence from yeast chromosome.
BAC vector Bacterial artificial chromosome is based on F plasmid of e coli. it contains genes for replication and maintenance of F factor, selectable marker and cloning sites.
Phagemid Composite structure made of bacteriophage and plasmid, used for carrying larger DNA sequences
Transposons as vector Unit of DNA which can move from one DNA molecule to another
Shuttle vectors These vector can replicate in both Eukaryotic and E coli that is these vectors contains two types of origin of replication and selectable marker genes one for Eukaryotic cell another for E coli
Tumor inducing (Ti) plasmid of agrobacterium tumefaciens Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as t DNA to transform normal plant cells into a tumor and direct these tumor cells to produce the Chemicals required by the pathogen.
Modification of Ti plasmid of agrobacterium tumefaciens Tumor inducing plasmid of agrobacterium tumefaciens has now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanism to deliver genes
Retroviruses These viruses in animals have the ability to transform normal cells into cancerous cells. These are also been designed and are now used to deliver desirable jeans into animal cells
Steps involved in RDT Isolation of DNA,fragmentation of DNA, separation and isolation of a desired DNA fragment, amplification of gene of interest using PCR, ligation of the DNA fragment into a vector,transferring the Recombinant DNA,culturing,extraction of the desired product
Isolation of DNA Cell extract is treated to remove all components except the DNA. RNA can be removed by treatment with ribonuclease and proteins can be removed by treatment with protease. Purified DNA ultimately precipitates out after the addition of chilled ethanol.
Fragmentation of DNA at specific locations by RE Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. Both vector and insert should be digested with compatible that is the same restriction enzyme.
How do we separate DNA fragments These fragments can be separated by a technique known as gel electrophoresis. Since DNA fragments are negatively charged they can be separated by forcing them to move towards the positive electrode anode under an electric field through a medium
Most commonly used medium for gel electrophoresis Agarose which is a natural polymer extracted from seaweeds
What is the sequence of separation of DNA fragments DNA fragments separate according to their size through seiving effect provided by the agarose gel. Hence smaller the fragments size, the father it moves.
How do we see separated DNA fragments The bright Orange coloured bands of DNA fragments can be visualised only after staining the DNA with a compound known as ethidium Bromide followed by exposure to UV radiation
What is elution The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece this step is known as elution.
What is PCR PCR stands for polymerase chain reaction. American biochemist Kary Mullis invented it. It enables the production or amplification of billions of copies of an original piece of DNA in a tube within minutes or hours.
A major limitation of early PCR method was that fresh DNA polymerase had to be added during every cycle. what eliminated the need? A thermostable DNA polymerase often referred to by its popular nickname taq polymerase, a heat resistant enzyme isolated from the bacterium thermus aquaticus eliminated the need to add fresh polymerase. Thermus aquaticus is a thermophilic bacteria.
Ingredients of polymerase chain reaction DNA polymerase, small amount of DNA to serve as the initial template, 4 deoxyribonucleotides to serve as the substrates for DNA polymerase few necessary ions and salts, and a pair of primers with exposed 3'-OH groups that will bind to the sequence
What are primers Small chemically synthesized oligonucleotides that are complementary to the regions of DNA
What is the use of primers DNA polymerases can only add new nucleotides to the 3'-OH end of a growing strand. They therefore required the presence of a primer to get started. In fact two primers are required, one to initiate replication of each of the two DNA strands
Steps of PCR reaction Single PCR reaction involves 3 temperature dependent steps Denaturation, annealing , primer extension (polymerization)
Denaturation Denaturation starting solution is heated, usually to 94 degree Celsius. The high temperatures break the hydrogen bonds between the two strands of the original DNA double helix, providing the necessary single-stranded templates
Annealing Reaction mixture is quickly cold, usually to somewhere between 50 degree Celsius and 60 degree Celsius, held for less than a minute which is enough time for the primers to bind to their complementary sequences on the single-stranded templates.
Primer extension Sample is next heated to 72 degree Celsius for some time during which time the DNA polymerase adds nucleotides to the primer, synthesizing a new DNA strand using only the template sequences that bind the primers
How do we make bacterial cells competent for DNA It is done by treating them with a specific concentration of a divalent cation such as calcium which increases the deficiency with which DNA enters the bacterium through pores in its cell wall. Calcium chloride improve DNA binding.
How do we do transformation that is a procedure through which a piece of DNA is introduced into a host bacterium Recombinant DNA can be forced into competent cells by incubating the cells with Recombinant DNA on ice. Followed by placing them briefly at 42 degree Celsius and then putting them back on ice. This enables the bacteria to take up the Recombinant DNA.
What is micro injection It is a method in which Recombinant DNA is directly injected into the nucleus of an animal cell.
What is biolistics or gene gun It is a method suitable for plants in which cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA
Define electroporation Short electrical impulses of high field strength are given. These increase the permeability of protoplast membrane by creating transient microscopic pores thus making entry of DNA molecules into the cells much easier.
What is a Recombinant protein If any protein encoding gene is expressed in a heterologous host it is called a Recombinant protein.
What is a bioreactor and which is the most commonly used one Fermenters, also known as bioreactors, are Chambers in which microorganisms are cultured in a liquid or solid medium. the most commonly used bioreactors are of stirring type.
What is the process and product of fermenter The process taking place in fermenters are fermentations. The product is either the cells themselves or some useful cell product. All operations must be carried out under trial conditions to avoid contamination of the culture
Types of fermentation Batch fermentation (closed system) continuous culture (open system)
What is batch fermentation In batch fermentation the conditions are set up and not changed from outside once the fermentation start. The process is stopped one sufficient product has been formed
Continuous culture system In this system, the used medium is drained out from one side, while fresh medium is added from other side to maintain the cells in their physiological in most active log or exponential phase. This type of culture method produces a larger Biomass.
What is downstream processing It is the name given to the stage after fermentation when the desired product is recovered and purified. These downstream processes include separation and purification of the desired product.
Created by: sherickp



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