Yeasts Flashcards
Yeast
single-celled eukaryotes, belong to fungi. Yeast likes to grow by fermentation of sugar (if in the presence of glucose)
Products of fermentation
Ethanol, carbon dioxide
Respiration
More energy efficient but slow
Fermentation as competition
Use up glucose quickly, poison microbes with ethanol, metabolism ethanol at leisure.
Chocolate and coffee
Yeast is also essential for fermentation of cacao and coffee beans. Yeast strains that hitchhiked with human migration are critical for the flavour of chocolate and coffee
Ethanol as biofuel
Biotech-> cellulosic ethanol from yeast fermentation
Fermentation - potentially carbon neutral Bioenergy
Yeast as model organism in research
Well characterise, small genome, rapid growth and breeding, easy to manipulate in almost any way you can imagine. Yeast genes homologous to human genes. Yeast as host for experiments with human proteins
S.cerevisiae life cycle
- the mating-type is controlled by genes at a single locus, MAT
haploids have either MATa allele or MATalpha allele
The MAT genes control mating-type via expression of pheromones and receptors
Wild strains can change mating type using a genetic switch
Diploids are heterozygous at MAT locus
isolating auxotrophic mutations in yeast
An auxotrophic mutant requires a specific nutrient that a wild-type cell can produce for itself
Wild-type yeast strains are prototrophic
Natural mutations are rare
So cells cannot grow on minimal medium- auxotrophic mutations.
Add defined nutrients to minimal medium, one at a time to see what nutrient they need
To speed up mutagenesis
Mutagenise liquid culture, dilate, plate on solid medium.
Cells are plated onto rich medium contains yeast extract, everybody can grow
Replica-plate to minimal medium
Tetrad analysis
Cross two different strains -> recombination, meiosis
Separate out four haploid spores: tetrad dissection
Germinate on rich medium
Replica-plate to minimal medium
Two mutations (if mutations are far apart- will segregate randomly with respect to each other. If mutations close together, crossovers with only rarely occur between the 2 mutations)
Molecular yeast genetics
Plasmids- common in wild yeast
Can be fused with an E.coli plasmid- add genetic markers to allow selection in either yeast or E.coli
‘shuttle vector’ (2 different host species)
How to clone a specific gene
Cut up genomic DNA from wild-type yeast and put pieces into plasmids
Test each plasmid for ability to complement auxotrophic lys2 mutant to find plasmid with wild-type LYS2 gene
Genomic library construction
Wild type genomic DNA restriction enzyme fragments. shuttle vector digested with restriction enzyme. Mix, ligate, transform into E.coli for amplifcation
Testing for candidate genes
1 step gene replacement. after finding a candidate clone, you want to make sure it really is the gene of interest.
Cut candidate gene with restriction enzyme.
Ligate in a known yeast marker gene.
Now cut out whole construct(insert), and transform into yeast selecting with the resistant drug
Yeast will attempt to repair the bare ends of the transformed DNA by recombining with its homologous sequence
Wild type gene gets replace by the mutated and marker gene construct.
