Biotechnology: Principles and Processes — Notes

Biotechnology (European Federation of Biotechnology): the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services. Two core techniques:

• Genetic engineering: altering the chemistry of genetic material (DNA/RNA) and introducing it into host organisms to change their phenotype.
• Bioprocess engineering: maintaining a sterile (microbial-contamination-free) ambience in chemical engineering processes so that only the desired microbe or eukaryotic cell grows, in large quantities.

Traditional hybridisation may carry undesirable genes along with desired ones; genetic engineering transfers only the desired genes.

First recombinant DNA (1972): Stanley Cohen and Herbert Boyer linked an antibiotic-resistance gene to a native plasmid of Salmonella typhimurium and transferred it into E. coli. A piece of DNA can multiply only when linked to an origin of replication (ori).

Three basic steps: (1) identify DNA with desirable genes; (2) introduce it into the host; (3) maintain it in the host and transfer it to progeny.

In 1963, two enzymes restricting bacteriophage growth in E. coli were isolated: one added methyl groups to DNA, the other cut it (restriction endonuclease). The first, Hind II, recognised a specific six-base-pair sequence (recognition sequence). More than 900 restriction enzymes from over 230 bacterial strains are now known.

Naming (EcoRI): E — genus Escherichia; co — species coli; R — strain RY 13; I — order of isolation. Restriction enzymes belong to nucleases: exonucleases remove nucleotides from the ends; endonucleases cut at specific positions within the DNA.

Palindromic sequences: each restriction endonuclease recognises a sequence that reads the same 5′→3′ on both strands, e.g. 5′-GAATTC-3′ / 3′-CTTAAG-5′. EcoRI cuts between G and A on each strand, a little away from the centre, leaving single-stranded overhangs (sticky ends) that form hydrogen bonds with complementary ends; DNA ligase joins them. Cutting the vector and source DNA with the same enzyme gives matching sticky ends.

Agarose gel electrophoresis: agarose (from seaweed) acts as a sieve; negatively charged DNA moves towards the anode; smaller fragments move farther; DNA stained with ethidium bromide shows orange bands under UV; bands are cut out and the DNA extracted (elution).

Vector features:

• Origin of replication (ori): starts replication; controls the copy number of linked DNA.
• Selectable marker: identifies transformants and eliminates non-transformants — genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin.
• Cloning sites: very few, preferably single, recognition sites for commonly used restriction enzymes.
• Small size.

pBR322: ori, rop, ampR and tetR; BamHI and SalI sites lie in tetR; PvuI and PstI in ampR. Insertional inactivation: an insert in tetR (e.g. at BamHI) makes recombinants ampicillin-resistant but tetracycline-sensitive. Alternative: insertion into the β-galactosidase gene; with a chromogenic substrate, non-recombinants give blue colonies and recombinants white.

Plant and animal vectors: Agrobacterium tumefaciens delivers T-DNA into plant cells; its Ti plasmid is disarmed for use as a vector. Retroviruses are disarmed to deliver genes into animal cells.

Competent hosts: DNA is hydrophilic and cannot cross membranes. Bacteria are treated with a divalent cation (calcium), incubated on ice with DNA, given a heat shock (42 °C) and returned to ice. Other methods: micro-injection (into animal cell nucleus), biolistics / gene gun (plant cells bombarded with gold or tungsten particles coated with DNA), disarmed pathogen vectors.

1. Isolation of DNA: break cells with lysozyme (bacteria), cellulase (plants) or chitinase (fungi); remove RNA with ribonuclease and proteins with protease; precipitate DNA with chilled ethanol (fine threads, spooling).
2. Cutting: restriction enzyme; progress checked by agarose gel electrophoresis; vector cut with the same enzyme.
3. Amplification by PCR: denaturation (high temperature separates strands), annealing (two primers bind), extension by thermostable Taq polymerase from Thermus aquaticus; copies = 2ⁿ after n cycles (about a billion after 30 cycles).
4. Ligation: gene joined to vector by DNA ligase → recombinant DNA.
5. Transformation and selection: recombinant DNA introduced into competent cells; cells grown on antibiotic medium so only transformants survive.
6. Obtaining the product: expressed gene gives a recombinant protein; large-scale growth in continuous culture (cells kept in log phase) in bioreactors (100–1000 L) providing optimal temperature, pH, substrate, salts, vitamins and oxygen. Stirred-tank bioreactor: curved-base cylinder; stirrer for mixing and oxygen availability; sterile air through a sparger; agitator, oxygen delivery, foam control, temperature and pH control, sampling ports.
7. Downstream processing: separation, purification, formulation with preservatives, clinical trials (drugs), strict quality control.