Biotechnology: Principles and Processes
Biotechnology uses live organisms or enzymes from organisms to make products and processes useful to humans. In the modern restricted sense it refers to processes using genetically modified organisms on a larger scale.
Principles (two core techniques)
- Genetic engineering: techniques to alter the chemistry of genetic material (DNA, RNA), introduce it into host organisms and change the host phenotype.
- Bioprocess engineering: maintaining a sterile (contamination-free) ambience to grow only the desired microbe/eukaryotic cell in large quantities to make antibiotics, vaccines, enzymes etc.
Unlike traditional hybridisation (which carries undesirable genes along), genetic engineering isolates and introduces only desirable genes. An alien DNA must be linked to an origin of replication to multiply in a host; making multiple identical copies = cloning. The first recombinant DNA was made by Cohen and Boyer (1972) by linking an antibiotic resistance gene to a Salmonella typhimurium plasmid, joined by DNA ligase, then cloned in E. coli.
Three basic steps of genetically modifying an organism: (i) identification of DNA with desirable genes; (ii) introduction into the host; (iii) maintenance in the host and transfer to progeny.
Tools of recombinant DNA technology
- Restriction enzymes: molecular scissors. First sequence-specific one = Hind II (recognises 6 bp). Naming e.g. EcoRI: genus (E), species (co), strain (R), order of isolation (I). Nucleases = exonucleases (cut from ends) + endonucleases (cut internally). They recognise palindromic sequences and cut away from the centre leaving single-stranded sticky ends (joined by DNA ligase).
- Gel electrophoresis: negatively charged DNA fragments move to the anode through agarose; smaller fragments move farther; stained with ethidium bromide + UV; bands cut out and recovered by elution.
- Cloning vectors need: ori (controls copy number), selectable marker (antibiotic resistance e.g. amp/tet/kan; or blue-white via beta-galactosidase insertional inactivation), and few/single cloning sites. Plant vector = Ti plasmid of Agrobacterium tumefaciens (disarmed); animal = disarmed retroviruses.
- Competent host: DNA is hydrophilic, so cells are made competent with a divalent cation (Ca2+); recombinant DNA forced in by heat shock (42 C). Other methods: micro-injection, biolistics/gene gun, disarmed pathogen vectors.
Processes of recombinant DNA technology (in sequence)
- Isolation of DNA - break cells with lysozyme/cellulase/chitinase; remove RNA (ribonuclease) and protein (protease); precipitate DNA with chilled ethanol.
- Cutting at specific locations with restriction enzymes (checked on agarose gel).
- Amplification by PCR - uses two sets of primers + DNA polymerase; thermostable polymerase from Thermus aquaticus; three steps per cycle (denaturation, primer annealing, extension); up to ~1 billion copies.
- Insertion into host - competent cells take up DNA; selectable marker (e.g. ampicillin resistance) selects transformants.
- Obtaining the gene product - protein in a heterologous host = recombinant protein; scaled up in bioreactors (100-1000 L), commonly stirred-tank type, kept in log phase by continuous culture.
- Downstream processing - separation, purification, formulation with preservatives, clinical trials, strict quality control.