Lesson 11 of 11 · 13 min
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Must-know facts
20 facts
- 1Two core techniques: genetic engineering and bioprocess engineering (sterile conditions for growing only the desired cells).
- 2First recombinant DNA: Cohen and Boyer, 1972, using an antibiotic-resistance gene and a plasmid of Salmonella typhimurium, cloned in E. coli.
- 3Three steps of genetic modification: identify DNA with the desired gene, introduce it into the host, maintain it and pass it to the progeny.
- 4Restriction endonuclease found in 1963; Hind II, the first sequence-specific one, five years later; now 900+ enzymes from 230+ strains.
- 5EcoRI: E = Escherichia, co = coli, R = strain RY 13, I = the first enzyme isolated from that strain.
- 6Exonucleases trim DNA from its ends; endonucleases cut within it.
- 7Palindrome: 5'-GAATTC-3' / 3'-CTTAAG-5'; cutting slightly off-centre leaves sticky ends that DNA ligase joins.
- 8Gel electrophoresis: DNA is negative and moves to the anode; the matrix is agarose from seaweed; smaller fragments move farther.
- 9Ethidium 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.
Common traps
Where marks are lost
Saying DNA runs towards the cathode.
DNA is negatively charged, so it moves towards the anode (positive electrode).
Thinking larger DNA fragments travel farther in the gel.
The agarose sieves the fragments, so the smallest ones move farthest from the well.
Reading blue colonies as recombinants.
Blue colonies have an intact β-galactosidase gene and no insert; recombinant colonies are colourless.
Expecting recombinants from pBR322 (insert at BamH I) to grow on tetracycline.
The insert inactivates tetR, so recombinants grow on ampicillin but die on tetracycline.
Reading the R in EcoRI as 'restriction'.
The R comes from the strain RY 13; the Roman numeral I gives the order of isolation.
Using lysozyme to break open plant or fungal cells.
Lysozyme is for bacteria; plant cells need cellulase and fungi need chitinase.
Giving the heat-shock temperature as 72°C or 94°C.
Heat shock for transformation is at 42°C, done briefly between two spells on ice.
Assuming any DNA polymerase works in PCR.
It must be thermostable, like Taq polymerase from Thermus aquaticus, to survive repeated denaturation.
Confusing downstream processing with the growth stage.
Downstream processing comes after biosynthesis: separation and purification, then formulation and quality control.
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.