Biotechnology: Principles and Processes — Short Notes
Biotechnology = using living organisms/cells or their components to make useful products or processes. EFB definition: integration of natural sciences and engineering for tech applications.
Two Core Techniques of Modern Biotechnology
- Recombinant DNA technology (genetic engineering) — introducing genes from one organism into another.
- Bioprocess engineering — sterile large-scale culture of cells for products.
Principles of Genetic Engineering
Three basic requirements to make recombinant DNA (rDNA):
- Identification & isolation of the desired gene.
- Introduction into a suitable host — needs a vector.
- Maintenance and multiplication in the host + selection of transformants.
Tools of rDNA Technology
Restriction enzymes ("molecular scissors")
- Restriction endonucleases cut DNA at specific sequences (usually 4–8 bp palindromes).
- First discovered: Hind II by H. Smith and W. Arber & D. Nathans (Nobel 1978).
- Named per organism: EcoRI = Escherichia coli R strain, first enzyme (I).
- Produce blunt or sticky ends; sticky ends have short single-stranded overhangs.
- DNA ligase seals cut fragments — used to join foreign DNA into vector.
Polymerase Chain Reaction (PCR)
- Amplifies specific DNA sequences million-fold.
- Requires: template DNA, primers, dNTPs, Taq polymerase (heat-stable, from Thermus aquaticus).
- Three steps per cycle:
- Denaturation at ~94-95°C — separates strands.
- Annealing at ~50-60°C — primers bind.
- Extension at ~72°C — polymerase adds nucleotides.
- 30 cycles → ~1 billion copies.
Vectors
- Plasmids — small circular DNA in bacteria; carry the gene into host.
- Common: pBR322; also bacteriophages.
- Features a vector must have:
- Origin of replication (ori).
- Selectable marker — often antibiotic resistance genes (e.g. amp^R, tet^R).
- Cloning sites — where restriction enzymes cut.
- Small size for easy manipulation.
Host organism (competent cell)
- Must be made competent to accept DNA.
- Chemical method: Ca²⁺ ions treatment + heat shock (42°C) + ice.
- Other methods: microinjection (animal), gene gun / biolistics (plants), disarmed pathogen vectors (Agrobacterium, retroviruses).
Recombinant DNA Technology Steps
- Isolation of DNA — cells broken with lysozyme (bacteria)/cellulase (plants)/chitinase (fungi); proteins with protease; RNA with ribonuclease; DNA precipitates with chilled ethanol.
- Fragmentation with restriction enzymes; run on agarose gel electrophoresis to check.
- Isolation of the desired fragment.
- Amplification by PCR or cloning into vector.
- Ligation into vector using DNA ligase → recombinant DNA.
- Insertion into host — competent cell + rDNA (transformation).
- Selection of transformed cells using markers (antibiotic resistance / insertional inactivation).
- Obtaining the foreign gene product — mass culture; downstream processing (separation, purification, quality control).
Bioreactors
- Large vessels (100–1000 L) for cell culture in controlled conditions.
- Stirred-tank reactor — most common; agitator provides mixing and O₂ transfer.
- Sparged stirred-tank reactor — sterile air blown in via a sparger.
Downstream Processing
Steps after fermentation to isolate the product:
- Separation of cells and supernatant.
- Purification (chromatography, precipitation).
- Formulation with preservatives / stabilisers.
- Stringent quality control tests.
Take-aways
- Genetic engineering = restriction enzymes + vectors + host + selection.
- PCR is the workhorse of DNA amplification — thanks to heat-stable Taq polymerase.
- Plasmid vectors like pBR322 must have ori, selection marker, cloning sites, and small size.
- Bioreactors and downstream processing convert lab-scale success into industrial products.
Biotechnology: Principles and Processes — Short Notes
Biotechnology = using living organisms/cells or their components to make useful products or processes. EFB definition: integration of natural sciences and engineering for tech applications.
Two Core Techniques of Modern Biotechnology
- Recombinant DNA technology (genetic engineering) — introducing genes from one organism into another.
- Bioprocess engineering — sterile large-scale culture of cells for products.
Principles of Genetic Engineering
Three basic requirements to make recombinant DNA (rDNA):
- Identification & isolation of the desired gene.
- Introduction into a suitable host — needs a vector.
- Maintenance and multiplication in the host + selection of transformants.
Tools of rDNA Technology
Restriction enzymes ("molecular scissors")
- Restriction endonucleases cut DNA at specific sequences (usually 4–8 bp palindromes).
- First discovered: Hind II by H. Smith and W. Arber & D. Nathans (Nobel 1978).
- Named per organism: EcoRI = Escherichia coli R strain, first enzyme (I).
- Produce blunt or sticky ends; sticky ends have short single-stranded overhangs.
- DNA ligase seals cut fragments — used to join foreign DNA into vector.
Polymerase Chain Reaction (PCR)
- Amplifies specific DNA sequences million-fold.
- Requires: template DNA, primers, dNTPs, Taq polymerase (heat-stable, from Thermus aquaticus).
- Three steps per cycle:
- Denaturation at ~94-95°C — separates strands.
- Annealing at ~50-60°C — primers bind.
- Extension at ~72°C — polymerase adds nucleotides.
- 30 cycles → ~1 billion copies.
Vectors
- Plasmids — small circular DNA in bacteria; carry the gene into host.
- Common: pBR322; also bacteriophages.
- Features a vector must have:
- Origin of replication (ori).
- Selectable marker — often antibiotic resistance genes (e.g. amp^R, tet^R).
- Cloning sites — where restriction enzymes cut.
- Small size for easy manipulation.
Host organism (competent cell)
- Must be made competent to accept DNA.
- Chemical method: Ca²⁺ ions treatment + heat shock (42°C) + ice.
- Other methods: microinjection (animal), gene gun / biolistics (plants), disarmed pathogen vectors (Agrobacterium, retroviruses).
Recombinant DNA Technology Steps
- Isolation of DNA — cells broken with lysozyme (bacteria)/cellulase (plants)/chitinase (fungi); proteins with protease; RNA with ribonuclease; DNA precipitates with chilled ethanol.
- Fragmentation with restriction enzymes; run on agarose gel electrophoresis to check.
- Isolation of the desired fragment.
- Amplification by PCR or cloning into vector.
- Ligation into vector using DNA ligase → recombinant DNA.
- Insertion into host — competent cell + rDNA (transformation).
- Selection of transformed cells using markers (antibiotic resistance / insertional inactivation).
- Obtaining the foreign gene product — mass culture; downstream processing (separation, purification, quality control).
Bioreactors
- Large vessels (100–1000 L) for cell culture in controlled conditions.
- Stirred-tank reactor — most common; agitator provides mixing and O₂ transfer.
- Sparged stirred-tank reactor — sterile air blown in via a sparger.
Downstream Processing
Steps after fermentation to isolate the product:
- Separation of cells and supernatant.
- Purification (chromatography, precipitation).
- Formulation with preservatives / stabilisers.
- Stringent quality control tests.
Take-aways
- Genetic engineering = restriction enzymes + vectors + host + selection.
- PCR is the workhorse of DNA amplification — thanks to heat-stable Taq polymerase.
- Plasmid vectors like pBR322 must have ori, selection marker, cloning sites, and small size.
- Bioreactors and downstream processing convert lab-scale success into industrial products.