Principles and Processes of Biotechnology Flashcards
Biotechnology
Biotechnology is the technique of using live organisms or enzymes from organisms to produce products and processes useful to mankind
Principles of biotechnology
- Genetic Engineering (alter the chemistry of DNA/RNA and introduce into host organisms)
- Maintenance of sterile environment for all genetic engineering processes
Cell lysis
Isolation of genomic DNA from bacteria/plant/animal cell
Tools of recombinant DNA technology
- Restriction enzymes
- Palindrome sequence
- Ligase
What are restriction enzymes
- They are molecular scissors/chemical scalpels
- Cut the DNA at specific sites into fragments
- E.g. EcoR1 (Escherichia coli) and Bam HI (Bacillus amyloliquifaciens)
Palindrome sequence
It is a sequence of base pairs that reads same in both strands when orientation of reading is kept the same
Ligase
Group of enzymes that carry out sealing, annealing or joining of DNA fragments
Cloning vectors
Plasmids
Bacteriophages
Features of a cloning vector
- Ability to replicate within a bacterial cell independent of the control of the chromosomal DNA
- Plasmids are lesser in a bacterial cell
- Phages are very large in number in a bacterial cell
Features of plasmids
- Small circular DNA molecules
- Self-replicating and exists in single or multiple copies
- Confers antibiotic resistance to the host cell
- Smaller in size than host (bacterial) chromosome
Phages
Usually have linear DNA molecules into which foreign DNA can be inserted
Cloning of a DNA fragment in a plasmid vector
- On a foreign DNA, the gene of interest is identified and isolated
- The cloning vector (plasmid) is also identified from the bacterial cell.
- Using the same restriction enzyme, both are cut
- The gene of interest is then ligated using ligase enzyme into the plasmid vector
- now it is called a recombinant plasmid vector
- It is then incorporated into the bacterial cell
- The bacterial cell is introduced into a culture medium
- Multiple copies of the desired gene of interest can be produced
Dolly
- Dolly was a female domestic sheep and the first mammal cloned from an adult somatic cell, using the process of nuclear transfer
- Birth: 5th July 1996
- Death: 14th February 2003
Write briefly the steps taken in cloning Dolly
- Donor egg cell + Mammary cell from sheep udder
- Enucleation of egg cell- so that it will read and duplicate the DNA of the donor cell
- Combine cells using an electric shock; the combined cell now has a single nucleus from the nuclear donor
- Combined cells divide to form a blastocyst (8 blastomeres)
- It is placed in the uterus of a surrogate ewe
- Blastocyst develops into a fetus and in ~5 months a lamb is born
Gene cloning
Molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
Features required to facilitate cloning
- Origin of replication (ori)
- Selective marker
- Cloning sites
i. antibiotic resistance selection
ii. Blue white selection - Vectors (small fragment- plasmid // large fragment- phage)
Antibiotic resistance selection
- Plasmids used in cloning contain an antibiotic resistance gene
- Thus, all bacteria are placed on an antibiotic plate to select for ones that took up a plasmid
- Bacteria without a plasmid die
- Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony
Processes of recombinant DNA technology
- Isolation of the genetic material (DNA)
- Cutting of DNA at specific sites- restriction enzymes (BamHI or EcoR1)
- Amplification of gene of interest using PCR (Polymerase CHain Reaction)
- Insertion of recombinant DNA into the host organism
- Obtaining the foreign gene product
- Downstream processing
Name the steps of PCR
- Denaturing
- Annealing
- Extension
Denaturing
Step 1 of PCR
The dsDNA target strand is separated at 95 degree celsius
Annealing
Step 2 of PCR
- Primers attach at 5’ end of each SsDNA
- Temperature is brought down to 55 degree celsius
Extension
Step 3 of PCR
- Taq polymerase extracted from thermophilic bacteria Thermus aquaticus is used.
- Temperature is raised to 72 degrees celsius for the enzyme to carry out polymerisation in the 5’-3’ direction of each strand
What happens after extension?
- After about 30 cycles of PCR about ~1 billion times (amplification) the target DNA sequence gets amplified
- The target gene is then cut using RE and inserted into a vector for further cloning procedure
YAC: Expansion, host cell, type of DNA molecule, Isolation of DNA, Cloning
VECTOR
Expansion: Yeast Artificial Chromosome
Host cell: Saccharomyces cerevisiae
Type of DNA molecule: Linear
Isolation of DNA: Difficult
Cloning: High cloning capacity as its insert can be up to ~1000kb in size
BAC: Expansion, host cell, type of DNA molecule, Isolation of DNA, Cloning
VECTOR
Expansion: Bacterial Artificial Chromosome
Host cell: Escherichia coli
Type of DNA molecule: Circular plasmid
Isolation of DNA: Easy
Cloning: Have less cloning capacity and its insert can be up to ~200kb in size
Methods of gene transfer using vector less systems
- Direct gene transfer
- Chemical method (transfection)
- Electroporation
- Micro-injection method
- Micro-projectile gene gun method
Direct gene transfer
Gene transfer using vector-less system
- Uptake of genes into the protoplast through the naked plasma membrane, followed by functional integration into the host genome
- Technique performed in tobacco plant, rice plant etc
Chemical method (Transfection)
Gene transfer using vector-less system
Process of inserting foreign DNA molecule into the host cell through a chemical DEAE-Dextran (Diethyl amino ethyl dextran)
Electroporation
Gene transfer using vector-less system
- Based on the principle of high voltage shock that induces cells to fuse
- Also induces cellular uptake of exogenous DNA from the surrounding solution
- It is an effective way of transforming E. coli cells containing plasmid DNAs larger than 100kb
Micro-injection method
Gene transfer using vector-less system
- DNA is injected under pressure into the protoplast/pollen/developing inflorescence
- Technique performed in tobacco plant, rye etc
Micro-projectile gene gun method
Gene transfer using vector-less system
- DNA is coated with tungsten or gold particles and projected by gene gun into the intact protoplasmic cells
- Technique performed in tobacco, soya bean etc
What is a bioreactor?
- bioreactors are used for commercial production of protein products.
- They provide optimal conditions for achieving the desired product (pH, temperature, substrate, salts, vitamins, and oxygen)
- Commonly used bioreactor is simple stirred tank bioreactor
Name the parameters on which the performance of bioreactors depends
- Agitation rate
- Oxygen transfer
- pH
- Temperature
- Foam production
Function of impellers in a bioreactor
Help in agitation/mixing of contents
Down-stream processing
- The desired product is separated from the debris and purified
- The desired recombinant product is then formulated with suitable preservatives and tested for quality and clinical trial is done (in case of drugs)
What is a sparged stirred tank bioreactor
- In sparged stirred tank bioreactor, sterile air is passed in the form of bubbles through the reactor
- The bubbles increase the surface area for oxygen transfer
HindII
isolated from Haemophilus influenzae
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