PDI1 yeast mutant酵母突变株 BioVector NTCC质粒载体菌种细胞基因保藏中心

PDI1 yeast mutant酵母突变株 BioVector NTCC质粒载体菌种细胞基因保藏中心 Standard Name
PDI1 1
Systematic Name
YCL043C
Aliases
MFP1 , TRG1
Feature Type
ORF , Verified
Description
Protein disulfide isomerase; multifunctional oxidoreductase of the ER lumen, essential for disulfide bond formation in secretory and cell-surface proteins, processing of non-native disulfide bonds; Ero1p activator; complexes with exomannosidase, Mnl1p to facilitate the recognition of misfolded glycoproteins and the trimming of glycan Man8GlcNAc2 to Man7GlcNAc2 on substrates, thereby accelerating ERAD; PDI1 has a paralog, EUG1, that arose from the whole genome duplication.
Paralog:EUG1
Introduction
The yeast knock out strains were systematically created using a PCR-based strategy. By means of two sequential PCR reactions - the first to incorporate the appropriate tags and confer the antibiotic resistance gene and the second to incorporate the mitotic recombination sites - each ORF was replaced with a KanMX cassette using homologous recombination. Each cassette contains a unique 20 base pair nucleotide sequence of DNA known as a "molecular barcode" allowing for parallel analysis. Also incorporated is a common set of flanking DNA tag sequences allowing amplification of the unique tags.
Strain Background Genotype
Mat A BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
Mat A BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
Het/Hom Dip BY4743 4741/4742
Homozygous diploids are in the BY4743 background unless 4730/4739 is indicated
Recovery
1. Obtain an YPD agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 30 o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. After 24-48hours, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.
References:
1. E. A. Winzeler et al., Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science. 285(5429), 901-906 (6 August 1999).
2. G. Giaever et al., Functional profiling of the Saccharomyces cerevisiae genome. Nature. 418, 387-391 (2002).
3. A. Wach, A. Brachat, R. Poehlmann, P. Philippsen, New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 10(13), 1793-1808 (December 1994).
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