Biotechnology: Principles and Processes: NEET notes

1. What biotechnology is

NCERT §9 (chapter introduction)

• Biotechnology covers techniques that use living organisms, or enzymes taken from them, to make products and run processes useful to people.
• In that broad sense, curd, bread and wine, all made with microbes, are biotechnology too.
• Today the word is used more narrowly for processes that use genetically modified organisms to do such things on a larger scale.
• Other techniques also count: IVF that produces a 'test-tube' baby, making a gene synthetically and putting it to use, building a DNA vaccine, and repairing a faulty gene.
• The European Federation of Biotechnology (EFB) gives a definition that covers both the traditional and the modern molecular kind: natural science brought together with organisms, cells, their parts and molecular analogues to yield products and services.

2. Principles of genetic engineering

NCERT §9.1

• Modern biotechnology rests on two core techniques. Genetic engineering changes the chemistry of the genetic material (DNA and RNA) and introduces it into host organisms, changing the host's phenotype.
• Bioprocess engineering keeps chemical-engineering processes sterile (free of microbial contamination), so that only the desired microbe or eukaryotic cell grows, in large quantities, to make products such as antibiotics, vaccines and enzymes.
• Traditional hybridisation in plant and animal breeding often carries undesirable genes along with the desired ones. Genetic engineering (recombinant DNA, gene cloning and gene transfer) lets us introduce only the one gene, or set of genes, that we want.
• A piece of foreign DNA put into an organism usually cannot multiply in its progeny cells. It multiplies only if it becomes part of a chromosome carrying the origin of replication, the sequence where replication begins.
• So foreign DNA is linked to an origin of replication, letting it replicate in the host. This making of many identical copies of a template DNA is cloning.
• The first recombinant DNA was made in 1972 by Stanley Cohen and Herbert Boyer. They cut an antibiotic-resistance gene out of a plasmid and joined it to a native plasmid of Salmonella typhimurium. A plasmid is circular, extra-chromosomal DNA that replicates on its own.
• Restriction enzymes act as molecular scissors that cut DNA at specific places. DNA ligase joins the cut ends. The plasmid is the vector that carries the foreign DNA, much as a mosquito carries the malarial parasite.
• Transferred into Escherichia coli, a close relative of Salmonella, the recombinant plasmid replicated using the host's DNA polymerase. This was called cloning the antibiotic-resistance gene in E. coli.
• Genetically modifying an organism takes three basic steps: identify the DNA carrying the desirable genes, introduce it into the host, and maintain it in the host and pass it on to the progeny.

3. Restriction enzymes

NCERT §9.2, §9.2.1

• Recombinant DNA technology needs these key tools: restriction enzymes, polymerase enzymes, ligases, vectors and a host organism.
• In 1963 two enzymes were isolated that restrict the growth of bacteriophage in E. coli. One adds methyl groups to DNA and the other cuts DNA; the cutting one was named restriction endonuclease.
• Five years later Hind II was isolated. It was the first restriction endonuclease whose action depended on a specific nucleotide sequence. It always cuts at a particular point, recognising a specific six-base-pair recognition sequence.
• More than 900 restriction enzymes are now known, isolated from over 230 bacterial strains, and each recognises a different sequence.
• Naming: the first letter comes from the genus and the next two from the species of the source prokaryote. EcoRI comes from Escherichia coli RY 13: 'R' is from the strain name, and the Roman numeral gives the order in which enzymes were isolated from that strain.
• Restriction enzymes are nucleases. Exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within it.
• An endonuclease scans the DNA for its recognition sequence, binds there, and cuts both strands at specific points in their sugar-phosphate backbones. Each recognises a specific palindromic sequence.
• A DNA palindrome reads the same on both strands when both are read in the same orientation. For example, 5'-GAATTC-3' pairs with 3'-CTTAAG-5'.
• The cut falls a little away from the centre of the palindrome, between the same two bases on each strand, leaving single-stranded overhangs called sticky ends. They hydrogen-bond with complementary cut ends, which helps DNA ligase join them.
• Source DNA and vector must be cut with the same restriction enzyme. The fragments then carry matching sticky ends that ligase can join end to end, forming recombinant DNA from different genomes.

4. Gel electrophoresis

NCERT §9.2.1 (separation and isolation of DNA fragments)

• Cutting DNA with restriction enzymes gives fragments, and these are separated by gel electrophoresis.
• DNA fragments carry a negative charge, so under an electric field they are forced through a matrix towards the anode (positive electrode).
• The matrix most used today is agarose, a natural polymer extracted from seaweeds.
• The gel acts as a sieve, so fragments separate (resolve) by size: the smaller the fragment, the farther it moves.
• Pure DNA cannot be seen in visible light without staining. The gel is stained with ethidium bromide and exposed to UV light, and the DNA shows as bright orange bands.
• Undigested DNA and digested DNA run in separate lanes, so the gel shows whether an enzyme has cut the sample.
• The wanted bands are cut out of the gel and the DNA is extracted from the gel piece. This step is elution, and the purified fragments are then joined to cloning vectors.

5. Cloning vectors

NCERT §9.2.2 (i)-(iii)

• Plasmids and bacteriophages replicate inside bacterial cells independently of the chromosomal DNA. Phages have very high copy numbers; some plasmids have one or two copies per cell and others 15-100, sometimes more.
• DNA linked to a plasmid or phage is multiplied to that vector's copy number. Modern vectors are engineered to make linking foreign DNA and selecting recombinants easy.
• Origin of replication (ori): the sequence where replication starts. Any DNA linked to it can replicate in the host, and ori also controls the copy number, so a high-copy ori is used to recover many copies.
• Selectable marker: it identifies and eliminates non-transformants and lets transformants grow. Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are useful markers for E. coli, since normal E. coli resists none of these.
• Cloning sites: the vector should have very few, preferably single, recognition sites for the common restriction enzymes. Several sites would cut it into many fragments and complicate cloning.
• pBR322, an E. coli vector, carries ori, ampR and tetR, rop (which codes for proteins involved in plasmid replication), and sites for Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I and Cla I.
• Foreign DNA ligated at the BamH I site inside tetR makes the recombinant lose tetracycline resistance. On ampicillin all transformants grow. Moved to tetracycline, recombinants fail while non-recombinants grow on both antibiotics.
• One resistance gene thus selects transformants, while the other, inactivated by the insert, picks out recombinants. Needing two plates makes this cumbersome.
• Easier: insert the DNA into the coding sequence of the enzyme β-galactosidase (insertional inactivation). With a chromogenic substrate, colonies without an insert turn blue; recombinant colonies stay colourless.

6. Plant and animal vectors, and competent hosts

NCERT §9.2.2 (iv), §9.2.3

• Methods of moving genes into plants and animals were learnt from bacteria and viruses. These pathogens deliver genes into eukaryotic cells and make the cells serve them.
• Agrobacterium tumefaciens, a pathogen of several dicots, delivers a piece of DNA called T-DNA. The T-DNA turns normal plant cells into a tumour that makes chemicals the pathogen needs. Retroviruses turn normal animal cells cancerous.
• The tumour-inducing (Ti) plasmid of Agrobacterium has been modified into a cloning vector. It no longer causes disease but still delivers chosen genes into many plants. Disarmed retroviruses deliver desirable genes into animal cells.
• DNA is hydrophilic and cannot pass through cell membranes, so bacteria must first be made competent to take it up.
• Cells are treated with a set concentration of a divalent cation, calcium for example, which helps DNA get in through pores in the cell wall more efficiently.
• The cells are incubated with recombinant DNA on ice, placed briefly at 42°C (heat shock), and returned to ice. This lets them take up the DNA.
• Micro-injection: recombinant DNA is injected directly into the nucleus of an animal cell.
• Biolistics (the gene gun), used for plants: tiny particles of gold or tungsten, coated with DNA, are fired into the cells at high velocity.
• Disarmed pathogen vectors transfer recombinant DNA into the cells they are allowed to infect.

7. Isolating and cutting DNA

NCERT §9.3, §9.3.1, §9.3.2

• The process runs in order: isolate DNA, cut it with restriction endonucleases, isolate the wanted fragment, ligate it into a vector, transfer it into the host, culture the host on a large scale, and extract the product.
• DNA must be pure, free of other macromolecules, before restriction enzymes can cut it. It lies within membranes, so cells are broken open to release it along with RNA, proteins, polysaccharides and lipids.
• Cell walls are broken with enzymes: lysozyme for bacteria, cellulase for plant cells, and chitinase for fungi.
• Genes lie on long DNA molecules wound around proteins such as histones. RNA is removed with ribonuclease and proteins with protease; other molecules have their own treatments.
• Adding chilled ethanol makes the purified DNA precipitate as fine threads in the suspension, which can be removed by spooling.
• Purified DNA is incubated with the restriction enzyme under that enzyme's optimal conditions. Agarose gel electrophoresis checks how far the digestion has gone, with DNA moving towards the anode. The vector DNA is cut the same way.
• The cut gene of interest and the cut vector are mixed and ligase is added, which gives recombinant DNA.

8. PCR

NCERT §9.3.3

• PCR, the polymerase chain reaction, synthesises many copies of a gene or DNA of interest in vitro.
• It uses two sets of primers and the enzyme DNA polymerase. Primers are short, chemically synthesised oligonucleotides complementary to regions of the DNA.
• Each cycle has three steps: denaturation of the double-stranded DNA, annealing of the primers, and extension of the primers.
• The polymerase extends the primers, using the nucleotides supplied and the genomic DNA as the template.
• Repeated many times, the process amplifies the DNA segment about a billion times, giving roughly 1 billion copies.
• Repeated cycles are possible because the DNA polymerase is thermostable. It is isolated from the bacterium Thermus aquaticus and stays active through the high temperature used to denature the DNA.
• The amplified fragment can then be ligated into a vector for further cloning.

9. Inserting DNA and getting the product

NCERT §9.3.4, §9.3.5

• Recipient cells made competent take up DNA from their surroundings.
• If recombinant DNA carrying an ampicillin-resistance gene enters E. coli, those cells become ampicillin-resistant.
• Spread on agar plates containing ampicillin, only the transformants grow and untransformed cells die. The ampicillin-resistance gene here is the selectable marker.
• Inside a bacterial, plant or animal cell, the foreign DNA in the vector is multiplied. Usually the final aim is a desirable protein, so the gene must be expressed, and it is expressed under suitable conditions.
• A protein made when a gene is expressed in a heterologous host is a recombinant protein.
• After the gene is cloned and expression is optimised, production is scaled up. Small laboratory cultures can be used to extract the protein and purify it by separation techniques.
• In a continuous culture system, spent medium drains out on one side as fresh medium flows in on the other. Cells stay in their most active log (exponential) phase, giving more biomass and a higher yield of protein.

10. Bioreactors and downstream processing

NCERT §9.3.5, §9.3.6

• Small cultures cannot give appreciable amounts of product, so bioreactors were developed to process large volumes of culture, 100-1000 litres.
• A bioreactor is a vessel in which raw materials are converted biologically into specific products or enzymes, using microbial, plant, animal or human cells.
• It keeps the conditions right for making the product: temperature, pH, substrate, salts, vitamins and oxygen.
• The most common type is the stirred-tank bioreactor: usually cylindrical or with a curved base to help mixing. The stirrer mixes the contents evenly and spreads oxygen through them.
• In a sparged stirred-tank bioreactor, sterile air bubbles are sparged (bubbled) through the culture.
• A bioreactor has an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system, and sampling ports for drawing off small volumes of culture from time to time.
• After the biosynthetic stage, the product goes through separation and purification, together called downstream processing.
• It is then formulated with suitable preservatives. Drugs must pass thorough clinical trials, and every product needs strict quality control testing. Both downstream processing and quality control vary from product to product.

Must-know facts

1. Two core techniques: genetic engineering and bioprocess engineering (sterile conditions for growing only the desired cells).
2. First recombinant DNA: Cohen and Boyer, 1972, using an antibiotic-resistance gene and a plasmid of Salmonella typhimurium, cloned in E. coli.
3. Three steps of genetic modification: identify DNA with the desired gene, introduce it into the host, maintain it and pass it to the progeny.
4. Restriction endonuclease found in 1963; Hind II, the first sequence-specific one, five years later; now 900+ enzymes from 230+ strains.
5. EcoRI: E = Escherichia, co = coli, R = strain RY 13, I = the first enzyme isolated from that strain.
6. Exonucleases trim DNA from its ends; endonucleases cut within it.
7. Palindrome: 5'-GAATTC-3' / 3'-CTTAAG-5'; cutting slightly off-centre leaves sticky ends that DNA ligase joins.
8. Gel electrophoresis: DNA is negative and moves to the anode; the matrix is agarose from seaweed; smaller fragments move farther.
9. Ethidium bromide with UV light shows orange DNA bands; cutting a band out and extracting its DNA is elution.
10. Vector features: ori (controls copy number), selectable marker, and single cloning sites.
11. pBR322: ori, ampR, tetR, rop; an insert at BamH I inactivates tetR.
12. Recombinants grow on ampicillin but not on tetracycline; non-recombinants grow on both.
13. Blue-white selection: an insert in the β-galactosidase gene gives colourless recombinant colonies; blue means no insert.
14. The Ti plasmid of Agrobacterium tumefaciens and disarmed retroviruses serve as vectors for plants and animals.
15. Competent cells: calcium (divalent cation), ice, heat shock at 42°C, ice again.
16. Micro-injection into the animal cell nucleus; biolistics fires gold or tungsten particles into plant cells.
17. Enzymes to break cells: lysozyme (bacteria), cellulase (plants), chitinase (fungi); ribonuclease and protease remove RNA and protein; chilled ethanol precipitates DNA, which is spooled.
18. PCR: denaturation, primer annealing, extension; Taq polymerase from Thermus aquaticus; about 1 billion copies.
19. Continuous culture keeps cells in the log phase; bioreactors hold 100-1000 litres; stirred-tank and sparged types are common.
20. Downstream processing is separation and purification, followed by formulation with preservatives, clinical trials for drugs, and quality control.

Common traps

Key terms

Biotechnology

Using living organisms or their enzymes to make products and processes useful to people.

Genetic engineering

Altering the chemistry of DNA or RNA and introducing it into a host to change its phenotype.

Bioprocess engineering

Keeping processes sterile so that only the desired cells grow, in large quantities.

Origin of replication (ori)

The sequence where replication starts; it also controls the copy number.

Plasmid

Circular extra-chromosomal DNA that replicates on its own in bacteria.

Vector

DNA such as a plasmid or phage that carries foreign DNA into a host.

Restriction endonuclease

An enzyme that cuts DNA within it at a specific recognition sequence.

Recognition sequence

The specific palindromic base sequence a restriction enzyme binds to and cuts.

Palindrome (DNA)

A sequence that reads the same on both strands when both are read in the same direction.

Sticky ends

Single-stranded overhangs left by a staggered cut, which pair with matching ends.

DNA ligase

The enzyme that joins cut DNA ends.

Gel electrophoresis

Separating DNA fragments by size as they move through agarose towards the anode.

Elution

Extracting DNA from a band cut out of the gel.

Selectable marker

A gene, often for antibiotic resistance, that lets only transformants grow.

Insertional inactivation

Loss of a gene's function when foreign DNA is inserted into it.

Competent cell

A bacterial cell treated, for example with calcium, so that it can take up DNA.

Biolistics

The gene gun: DNA-coated gold or tungsten particles fired into plant cells.

PCR

Polymerase chain reaction: cycles of denaturation, annealing and extension that copy DNA in vitro.

Bioreactor

A vessel of 100-1000 litres where cells turn raw materials into products under controlled conditions.

Downstream processing

Separation and purification of the product after the biosynthetic stage.