Lesson 11 of 11 · 13 min
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Must-know facts
20 facts
- 01Two core techniques: genetic engineering and bioprocess engineering (sterile conditions for growing only the desired cells).
- 02First recombinant DNA: Cohen and Boyer, 1972, using an antibiotic-resistance gene and a plasmid of Salmonella typhimurium, cloned in E. coli.
- 03Three steps of genetic modification: identify DNA with the desired gene, introduce it into the host, maintain it and pass it to the progeny.
- 04Restriction endonuclease found in 1963; Hind II, the first sequence-specific one, five years later; now 900+ enzymes from 230+ strains.
- 05EcoRI: E = Escherichia, co = coli, R = strain RY 13, I = the first enzyme isolated from that strain.
- 06Exonucleases trim DNA from its ends; endonucleases cut within it.
- 07Palindrome: 5'-GAATTC-3' / 3'-CTTAAG-5'; cutting slightly off-centre leaves sticky ends that DNA ligase joins.
- 08Gel electrophoresis: DNA is negative and moves to the anode; the matrix is agarose from seaweed; smaller fragments move farther.
- 09Ethidium bromide with UV light shows orange DNA bands; cutting a band out and extracting its DNA is elution.
- 10Vector features: ori (controls copy number), selectable marker, and single cloning sites.
- 11pBR322: ori, ampR, tetR, rop; an insert at BamH I inactivates tetR.
- 12Recombinants grow on ampicillin but not on tetracycline; non-recombinants grow on both.
- 13Blue-white selection: an insert in the β-galactosidase gene gives colourless recombinant colonies; blue means no insert.
- 14The Ti plasmid of Agrobacterium tumefaciens and disarmed retroviruses serve as vectors for plants and animals.
- 15Competent cells: calcium (divalent cation), ice, heat shock at 42°C, ice again.
- 16Micro-injection into the animal cell nucleus; biolistics fires gold or tungsten particles into plant cells.
- 17Enzymes to break cells: lysozyme (bacteria), cellulase (plants), chitinase (fungi); ribonuclease and protease remove RNA and protein; chilled ethanol precipitates DNA, which is spooled.
- 18PCR: denaturation, primer annealing, extension; Taq polymerase from Thermus aquaticus; about 1 billion copies.
- 19Continuous culture keeps cells in the log phase; bioreactors hold 100-1000 litres; stirred-tank and sparged types are common.
- 20Downstream processing is separation and purification, followed by formulation with preservatives, clinical trials for drugs, and quality control.
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Key terms
20 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.