yeast transformation uptake of foreign dna by a cell changes its phenotype
TRANSCRIPT
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
Electron micrograph by Christopher BuserUsed with permission(Cell Image Library:www.celllibrary.org)
"Fuzziness" is characteristic of polysaccharides
Cell wall is an extensively cross-linked 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
Chemicals used to transform yeast include:
Polyethylene glycolPossible effects on membrane structure
May help DNA adhere to the cell wall
Lithium acetateMonovalent cations generally enhance uptake of DNA
Single-stranded DNASaturates non-specific binding sites for DNA in cell wall
May provide protection from nucleasesDNA has been boiled and quick-chilled to make it single-
stranded
ura3
Plasmid transformation
ura3
URA3
URA3URA3
URA3
URA3
URA3
ura3
URA3URA3 URA3
URA3
Cells with weakened cell walls are incubated with plasmids
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?
pBG1805 (6573 bp)
S. cerevisiae ORF
pYES2.1 (5886 bp)
S. pombe ORF or LacZ
Our plasmids carry the S. cerevisiae URA3 gene and its promoter
Yeast normally synthesize UMP de novo*
*de novo – no external precursors are required
Ura3p
glutamine UMPorotidine- 5’-phosphate
UMP is synthesized from glutamine by a multi-step pathway
Ura3p (orotidine-5’-phosphate decarboxylase) catalyzes the final step in UMP synthesis
glutamine UMPorotidine- 5’-phosphate X
BY4742 genotype:MATa his3∆1 leu2∆0 ura3∆0 lys2∆0
uracil
uracil
salvage enzymes convert uracil to UMP
BY4742 strain has a deletion of the URA3 gene
BY4742 requires uracil to grow
ura3
URA3URA3 URA3
URA3
Ura3p
Ura3p
Ura3p
Ura3p
Complementation allows transformed cells to grow in the absence of uracil
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?
pBG1805 (6573 bp)
S. cerevisiae ORF
pYES2.1 (5886 bp)
S. pombe ORF or LacZ
Our experimental question relates to the MET genes carried by the plasmid. Expression is controlled by the GAL1
promoter
master plate
orientation marker
master plate media selective media plates
Step 1 - transfer colonies to sterile velveteen with gentle tapping
Step 2 – transfer colonies to various media
Step 3 - Incubate plates at 30˚C
Step 4 – Score plates for growth
Replica plating allows rapid screening of colonies for multiple phenotypes
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?
MET
Linear DNA in transformation is less efficient than plasmid transformation, but generates stable strains
KANR
KANR
KANR
KANR
KANR
MET KANR
KANR
Homologous recombination
Selection on kanamycin plates(no complementation involved)
Stable strain: KANR gene is integrated into the chromosome