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Biotechnolgy - Principles & Processes - Coggle Diagram
Biotechnolgy - Principles & Processes
EFB
PRINCIPLES OF BIOTECHNOLOGY
1.Genetic engineering
2. Bioprocess engineering
Cloning
Alien DNA + Ori = multiply in host cell
first recombinant
DNA
AB res gene + a native plasmid of
Salmonella typhimurium
= rDNA into
E.coli
E.coli
gets AB resistance
Stanley Cohen and
Herbert Boyer - 1972
TOOLS OF RECOMBINANT DNA TECHNOLOGY
1. Restriction Enzymes
1963, the two enzymes restrict the growthof bacteriophage in E. coli isolated 1. Add methyl groups to DNA 2. Cut DNA
first restriction endonuclease
–Hind II - six base pairs - 5'CAGCTG3'
more than 900 REs isolated from over 230 strains of bacteria
Nomenclature, Types, Restriction site - Palindromic, Sticky ends, DNA ligase
Gel electrophoresis
- Agarose (polymer from sea weeds) - Cathode to Anode
Separation based on size, the smaller the fragment size, the farther it moves
2. Cloning Vectors
Plasmids and bacteriophages - replicate in host independent of the control of chromosomal DNA
bacteriophages - high copy number per cell
Copy nos range = 1 to 15-100 per cell
A.Ori
Initiate replication
B. Selectable Marker
genes encoding resistance to antibiotics such as ampicillin, chloramphenicol,
tetracycline or kanamycin
C. Cloning sites
more than one recognition sites within the vector will generate several fragments - complicates cloning
Insertional inactivation
Presence of insert - White colored colonies, Absence - blue colonies
pBR322 - 2 AB res genes - one for cloning, other for selection
D. Vectors
Ti plasmid of Agrobacterium tumifaciens
Pathogen of dicots
Retroviral vectors
3. Competent Host
DNA - Hydrophilic
Treatment with divalent Cation - Ca2+
- increases the efficiency of DNA entry through pores
2. micro-injection -
rDNA is directly injected into nucleus of animal cell
**
3. Biolistics or gene gun -
high velocity micro-particles of gold or tungsten
coated with DNA - Suitable for Plants
4. Disarmed pathogen’ vectors
Incubation in ice - followed by heat shock at 42C
PROCESSES OF rDNA TECHNOLOGY
1. Isolation of the Genetic Material (DNA)
2. Cutting of DNA at Specific Locations
3. Amplification of Gene of Interest using PCR
4. Insertion of rDNA into the Host Cell/Organism
5. Foreign Gene Product
6. Downstream Processing
separation and purification
suitable preservatives
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Heterlogous host - rec protein
Bioreactors - raw materials
biologically converted to specific products
Optimum - temperature, pH, substrate, salts, vitamins,oxygen
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