Yeast transformation
Uptake of foreign DNA by a cell
changes its phenotype
What is transformation?
How is complementation used to select transformed strains?
How is replica plating used to screen for multiple phenotypes
in a strain?
What transformation strategy did the Saccharomyces Gene
Deletion Project use to generate the metX::KANR strains?
During transformation, DNA must cross the formidable yeast
cell wall
"Fuzziness" is characteristic of
polysaccharides
Cell wall is an extensively crosslinked network of proteins and
polysaccharides
Investigators have EMPIRICALLY
developed conditions for
transforming yeast
Cells are treated with chemicals
and submitted to a mild heat shock
Electron micrograph by Christopher Buser
Used with permission
(Cell Image Library:www.celllibrary.org)
Chemicals used to transform yeast include:
Polyethylene glycol
Possible effects on membrane structure
May help DNA adhere to the cell wall
Lithium acetate
Monovalent cations generally enhance uptake of DNA
Single-stranded DNA
Saturates non-specific binding sites for DNA in cell wall
May provide protection from nucleases
DNA has been boiled and quick-chilled to make it single-stranded
Plasmid transformation
Cells with weakened cell walls
are incubated with plasmids
ura3
URA3
URA3
URA3
URA3
URA3
ura3
URA3
URA3
ura3
URA3
URA3
URA3
Transformed cells are
isolated on selective
media, where they
recover and grow again
Cells must be continuously maintained on selective media to maintain the plasmids
What is transformation?
How is complementation used to select transformed strains?
How is replica plating used to screen for multiple phenotypes
in a strain?
What transformation strategy did the Saccharomyces Gene
Deletion Project use to generate the metX::KANR strains?
Our plasmids carry the S. cerevisiae URA3 gene and its promoter
S. cerevisiae
ORF
pBG1805 (6573 bp)
S. pombe
ORF or LacZ
pYES2.1 (5886 bp)
Yeast normally synthesize UMP de novo*
Ura3p
glutamine
orotidine5’-phosphate
UMP
UMP is synthesized from glutamine by a multi-step pathway
Ura3p (orotidine-5’-phosphate decarboxylase) catalyzes the
final step in UMP synthesis
*de novo – no external precursors are required
BY4742 strain has a deletion of the URA3 gene
orotidine5’-phosphate
glutamine
X
salvage enzymes convert
uracil to UMP
BY4742 genotype:
MATa his3∆1 leu2∆0 ura3∆0 lys2∆0
uracil
BY4742 requires uracil to grow
uracil
UMP
Complementation allows transformed cells to grow in the absence of uracil
URA3
Ura3p
ura3
Ura3p
URA3
URA3
URA3
Ura3p
Ura3p
Plasmid-encoded Ura3p complements ura3 deficiency in BY4742
Cells must be continually propagated in selective media to retain the plasmid
What is transformation?
How is complementation used to select transformed strains?
How is replica plating used to screen for multiple phenotypes
in a strain?
What transformation strategy did the Saccharomyces Gene
Deletion Project use to generate the metX::KANR strains?
Our experimental question relates to the MET genes carried by
the plasmid. Expression is controlled by the GAL1 promoter
S. cerevisiae
ORF
pBG1805 (6573 bp)
S. pombe
ORF or LacZ
pYES2.1 (5886 bp)
Replica plating allows rapid screening of colonies for multiple phenotypes
Step 4 – Score plates for growth
master plate
Step 3 - Incubate plates at 30˚C
master plate media
selective media plates
Step 2 – transfer colonies to various media
orientation
marker
Step 1 - transfer
colonies to sterile
velveteen with gentle
tapping
What is transformation?
How is complementation used to select transformed strains?
How is replica plating used to screen for multiple phenotypes
in a strain?
What transformation strategy did the Saccharomyces Gene
Deletion Project use to generate the metX::KANR strains?
Linear DNA in transformation is less efficient than plasmid
transformation, but generates stable strains
KANR
MET
KANR
KANR
KANR
KANR
MET
KANR
Homologous recombination
Selection on kanamycin plates
(no complementation involved)
Stable strain:
KANR gene is
integrated into
the chromosome
KANR