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Cloning beyond E.coli lecture notes
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Cloning beyond E.ColiCloning in other bacteria:
E.coli is the principle workhouse of the molecular biologist.
Plasmid (and a few phage) vectors exist for other bacteria:
- Streptomyces
- Bacillus
- Pseudomonas
Including broad host-range vectors:
- Will work across several bacterial species
- Horizontal gene transfer
Essentially, similar in mode of action to E.coli vectors
Cloning in yeast:
Saccharomyces cerevisiae
- Used in brewing & baking
- Biotechnology – pharmaceuticals
- Model eukaryotic organism
Vectors based on 2μm plasmid
2 μm plasmid:
Contains several genes:
- REP1 and REP2 involved in replication (chromosomal
genes also involved)
- FLP flips the gene order
- D function unkown
ori is essential for replication (replication origin)
But no selectable marker
LEU2 as a selectable marker:
Use an auxotrophic yeast host that has a mutant
leu2 gene
, - Cannot synthesise leucine
- Must be grown on medium containing leucine
2 μm plasmid vector contains LEU2 gene
- Transformed cells plated on minimal medium (without
leucine)
- Functional LEU2 in plasmid will complement mutant leu2
gene in host
- Enabling growth of transformed yeast cells
YEp13 – yeast episomal vector:
Amalgam of PBR322, LEU2 gene and ori from 2 μm
Shuttle vector – can be used in E.coli (due to
ampicillin resistance gene & tetracycline resistance
gene – selectable markers) and yeast (LEU2 is yeast
selectable marker)
Can replicate as independent plasmid in yeast
Can integrate into yeast genome via homologous
recombination between LEU2 and leu2 – hence
episomal
Use as an E.coli-yeast shuttle vector:
Homologous recombination:
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