• Techniques to alter the chemistry of genetic material (DNA and RNA)
• Introduce these into host organisms
• Change the phenotype of the host organism

• Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes
• Enables growth of only the desired microbe/eukaryotic cell in large quantities
• Used for manufacture of — antibiotics, vaccines, enzymes, etc.

• Isolates and introduces only desirable genes
• Without introducing undesirable genes
• Includes — recombinant DNA, gene cloning, gene transfer

• Alien DNA mostly cannot multiply in progeny cells
• If it integrates into the genome → multiplies and is inherited with host DNA
• Becomes part of a chromosome which can replicate
• Chromosome has origin of replication → responsible for initiating replication
• Alien DNA must be linked with origin of replication to replicate in host
• This process = Cloning (making multiple identical copies of template DNA)

• Linked antibiotic resistance gene with native plasmid of Salmonella typhimurium
• Isolated antibiotic resistance gene by cutting DNA from a plasmid
• Cutting possible by 'molecular scissors' → restriction enzymes
• Cut DNA linked with plasmid DNA → plasmid acts as vector
• Linking done by enzyme DNA ligase → joins ends of cut DNA
• New circular autonomously replicating DNA created in vitro = Recombinant DNA
• Transferred into Escherichia coli → replicated using host's DNA polymerase
• This was called cloning of antibiotic resistance gene in E. coli

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 to its progeny

• Two enzymes isolated responsible for restricting growth of bacteriophage in E. coli
• One added methyl groups to DNA
• Other cut DNA → called restriction endonuclease

• Isolated and characterised 5 years later
• Recognised a specific sequence of six base pairs
• This specific sequence = recognition sequence for Hind II
• Today → more than 900 restriction enzymes isolated from over 230 strains of bacteria

• 1st letter → from Genus
• 2nd and 3rd letters → from species
• Letter after → derived from name of strain
• Roman numbers → indicate order of isolation from that strain

Restriction enzymes belong to larger class → Nucleases

• Inspects the length of a DNA sequence
• Finds its specific recognition sequence
• Binds to DNA and cuts each of the two strands at specific points in their sugar-phosphate backbones
• Recognises a specific palindromic nucleotide sequence

A sequence of base pairs that reads same on both strands when orientation of reading is kept the same.

• Restriction enzymes cut a little away from the centre of palindrome sites
• But between the same two bases on opposite strands
• Leaves single stranded overhanging portions = sticky ends
• Named so because they form hydrogen bonds with complementary cut counterparts
• Stickiness facilitates the action of DNA ligase

• DNA fragments are negatively charged → move towards anode under electric field
• Matrix used → Agarose (natural polymer extracted from sea weeds)
• Fragments separate according to size (sieving effect)
• Smaller fragment → moves farther
• DNA stained with ethidium bromide → exposed to UV radiation
• Shows bright orange coloured bands of DNA

If alien DNA is linked with plasmid/bacteriophage → its numbers multiply equal to the copy number of that vector.

• Sequence from where replication starts
• Any DNA linked to this → can replicate within host cells
• Also controls the copy number of linked DNA
• For many copies → clone in vector with high copy number ori

• Helps in identifying and eliminating non-transformants
• Selectively permits growth of transformants
• Transformation = procedure through which a piece of DNA is introduced in a host bacterium
• Useful markers for E. coli → resistance to: Ampicillin, Chloramphenicol, Tetracycline, Kanamycin
• Normal E. coli cells → do not carry resistance against any of these

• Based on colour production using chromogenic substrate
• Recombinant DNA inserted within coding sequence of enzyme β-galactosidase
• Inactivates the gene → called insertional inactivation

• Vector must have very few, preferably single recognition sites for restriction enzymes
• More than one recognition site → generates several fragments → complicates gene cloning
• Ligation of alien DNA at restriction site present in one of the two antibiotic resistance genes

• Treat with specific concentration of divalent cation (e.g., Calcium)
• Increases efficiency of DNA entry through pores in cell wall

1. Incubate cells with recombinant DNA on ice
2. Place briefly at 42°C → Heat shock
3. Put back on ice → Bacteria take up recombinant DNA

1. Isolation of DNA
2. Fragmentation of DNA by restriction endonucleases
3. Isolation of a desired DNA fragment
4. Ligation of the DNA fragment into a vector
5. Transferring the recombinant DNA into the host
6. Culturing the host cells in a medium at large scale
7. Extraction of the desired product

• Nucleic acid is the genetic material of all organisms without exception
• In majority of organisms → DNA
• DNA must be in pure form, free from other macromolecules for restriction enzymes to cut it
• DNA is enclosed within membranes → cell must be broken open
• Breaking open releases → DNA, RNA, proteins, polysaccharides, lipids

• Genes located on long DNA molecules interwined with proteins (histones)
• RNA removed → by treatment with ribonuclease
• Proteins removed → by treatment with protease
• Purified DNA → precipitates out after addition of chilled ethanol
• Seen as fine threads in the suspension

• Performed by incubating purified DNA with restriction enzyme at optimal conditions
• Agarose gel electrophoresis → used to check progression
• DNA is negatively charged → moves towards positive electrode (anode)
• Same process repeated with vector DNA

• Source DNA and vector DNA cut with same specific restriction enzyme
• Cut gene of interest + cut vector are mixed
• Ligase added → results in preparation of recombinant DNA

PCR = Polymerase Chain Reaction

• Multiple copies of gene/DNA of interest synthesised in vitro
• Uses two sets of primers → small chemically synthesised oligonucleotides complementary to regions of DNA
• Enzyme DNA polymerase extends the primers using nucleotides provided and genomic DNA as template
• Repeated many times → DNA segment amplified to approximately 1 billion copies

Amplified fragment can be ligated with a vector for further cloning.

• Recipient cells made 'competent' → then take up DNA from surroundings
• Recombinant DNA bearing ampicillin resistance gene transferred into E. coli
• Host cells become ampicillin-resistant (transformed)
• Transformed cells spread on agar plates containing ampicillin
• Only transformants grow → untransformed cells die
• Ampicillin resistance gene here = selectable marker

• Alien DNA inserted into cloning vector → transferred into host → gets multiplied
• Ultimate aim → produce a desirable protein
• Recombinant DNA must be expressed
• If protein encoding gene expressed in a heterologous host → called recombinant protein

• Cells grown in laboratory
• Desired protein extracted and purified using different separation techniques

• Used medium drained from one side
• Fresh medium added from other side
• Maintains cells in log/exponential phase
• Produces larger biomass → higher yields of desired protein

• Large volumes — 100–1000 litres of culture processed
• Vessels where raw materials biologically converted into specific products using microbial, plant, animal or human cells
• Provides optimal conditions → temperature, pH, substrate, salts, vitamins, oxygen

• Usually cylindrical or with curved base → facilitates mixing
• Stirrer → ensures even mixing and oxygen availability
• Alternatively → air can be bubbled through reactor

After completion of the biosynthetic stage, the product is subjected through a series of processes before it is ready for marketing as a finished product.

• Product formulated with suitable preservatives
• Must undergo thorough clinical trials (in case of drugs)
• Strict quality control testing required for each product
• Downstream processing and quality control testing vary from product to product