Yap 180 gene in Yeast. what media will you need to grow and how to grow your yea
ID: 259080 • Letter: Y
Question
Yap 180 gene in Yeast.
what media will you need to grow and how to grow your yeast?
Do you need to test it under different conditions such as different temperature, sensitive to antifungals, UW or chemicals?
This is a method that i need to find information.
METHODS
Yeast strains and media
Yeast strains (listed in supplementary material Table S1) were transformed with plasmids carrying the different chimeric constructs under the GAL10 promoter and were grown overnight on dextrose medium (0.67% yeast nitrogen base, 2% dextrose) supplemented with 0.67% casamino acids or with some of the following amino acids: 20 mg/l histidine (H), 20 mg/l lysine (K) and 60 mg/l leucine (L). Upon reaching exponential phase (OD600=1), the cells were placed in galactose medium supplemented with either 0.67% casamino acids or HKL to induce the expression of chimeric proteins. After 6 hours, cells were either collected to obtain total cell extracts or were observed under epifluorescence.
The ?yap1801/1802 strain was constructed by mating the two simple deleted strains ?yap1801 and ?yap1802. The mating type of the ?yap1801 strain was changed by transforming the strain with a plasmid carrying the HO gene. The two strains were then crossed by patching them mixed on a selective medium. After verifying mating by microscopy, the diploid strain was patched onto a sporulation medium. After 3 days of growth, tetrads were dissected and the resistance of the spores to G418 was tested. The G418-resistant spores from the double recombined tetrads were kept as ?yap1801/1802 strains. To lose the plasmid carrying the HO gene,?yap1801/1802 was then plated onto rich medium containing 5FOA.
Oligonucleotides are listed in supplementary material Table S2. The A?1–42 sequence was amplified by PCR from pSG5-APP (a kind gift from Agnès Hémar) using oligonucleotide 792, which introduces a BamHI restriction site at the 5? end of the fragment and an ATG codon at the beginning of the A?1–42sequence, and oligonucleotide 794. The PCR fragment was then inserted into the plasmid pYecHetsYGFP (Couthouis et al., 2009), which had been previously linearized by BamHI using a gap repair method (Orr-Weaver and Szostak, 1983). The pYe?A?YGFP and pYe?A?ARCYGFP plasmids were constructed by cloning a synthetic sequence in a BamHI-BstXI-digested pYeA?YGFP plasmid. These synthetic sequences, made by GeneScript, were composed of BamHI restriction site followed by ?-factor prepro sequence, A? wild-type or arctic mutant coding sequence, the 5? end of the GFP sequence, and a BstXI restriction site. The pYeA?ARCYGFP plasmid was created by overlapping PCR using pYe?A?ARCYGFP as a template (with oligonucleotides 705, 706, 859 and 860). This allowed the amplification of a PGAL-A?ARC-GFP fragment, which was introduced by a gap repair method into a BamHI-BstXI-digested pYe?A?ARCYGFP plasmid. Similarly, PGAL-?-factor prepro-GFP and PGAL-GFP sequences were created by overlapping PCR using pYe?A?YGFP and pYeA?YGFP as templates (with oligonucleotides 705, 706, 856, 857 and 858). The fragments were respectively inserted into pYe?A?YGFP and pYeA?GFP BamHI-BstXI-digested plasmids. Each of these plasmids is a multicopy yeast-expression plasmid with the URA3 selectable marker and a GAL10 promoter in a pYeHFN2U backbone (Cullin and Minvielle-Sebastia, 1994). CALM long splice variant (CALM-L; GenBank ID BC011470) and short splice variant (CALM-S; GenBank ID BC021491) from the American Tissue Culture Collection (ATCC) were amplified by PCR using oligonucleotide 951, which introduced a BamHI site at the 5? end of the cDNA and oligonucleotide 952 which introduced a NotI site at the 3? end of the cDNA. After digestion by BamHI and NotI, the PCR fragment was inserted into the pYeHFN2L plasmid. This plasmid is a multicopy yeast-expression plasmid with an LEU2 selectable marker and a GAL10promoter (Cullin and Minvielle-Sebastia, 1994).
Spotting assay
All spotting assays were performed under the same conditions. Tenfold serial dilutions starting with an equal number of cells (1 OD; where 1=600 nm) were performed in sterile water. Spotting assays were derived from a pool of three independent fresh transformants. Drops of 10 ?l were then plated onto the appropriate SD or SG medium.
Fluorescence microscopy
Cells were washed in water and resuspended in medium. An Axioskop 2 plus (Zeiss) fluorescence microscope was coupled with an AxioCam (Zeiss) black and white camera. The following filters were used: LP-GFP (GFP) and N3 (RFP).
Protein extraction, deglycosylation and western blotting
The alkaline lysis method was used for protein extraction. Briefly, 5 OD units of yeast cells in exponential growth phase were permeabilized with 500 ?l of 0.185 M NaOH, and 0.2% ?-mercaptoethanol. After 10 minutes of incubation on ice, trichloroaceticacid (TCA) was added to obtain a final concentration of 5%, and the samples were incubated for an additional 10 minutes on ice. Precipitates were then collected by centrifugation at 13,000 g for 5 minutes. Pellets were dissolved in 35 ?l of dissociation buffer (4% sodium dodecyl sulfate, 0.1 M Tris-HCl pH 6.8, 4 mM EDTA, 20% glycerol, 2% 2-mercaptoethanol and 0.02% Bromophenol Blue) and 15 ?l of 1 M Tris-base. For deglycosylation assays, pellets were suspended in 20 ?l glycoprotein denaturing buffer (Biolabs), incubated for 10 minutes at 100°C, and transferred for a few minutes at 4°C. We then added 5 ?l 10× G7 reaction buffer, 5 ?l 10% NP40 and 5 ?l deglycosylation enzyme cocktail (PNGase F, 500,000 U/ml; endo-?-N-acetylgalactosaminidase, 400,000,000 U/ml; neuraminidase, 50,000 U/ml; ?1-4 galactosidase, 8000 U/ml; and?-N-acetylglucosaminidase, 4000 U/ml) to obtain a volume of 50?l. The samples were then incubated for 4 hours at 37°C before adding 15 ?l of sample buffer.
Yeast proteins were incubated for 5 minutes at 100°C and separated by SDS-PAGE in a 12% polyacrylamide gel. Proteins were electrically transferred onto nitrocellulose membranes (Optitran BA-S83; Schleicher and Schuell) in the presence of transfer buffer (39 mM glycine, 48 mM Tris-base, 2% EtOH and 0.037% SDS) and were probed with monoclonal anti-GFP antibodies (Sigma) or anti-A? (Tebu) antibodies. Peroxidase-conjugated anti-mouse antibodies (Sigma) were used as secondary antibodies. Binding was detected with the SuperSignal reagent (Pierce) and the VersaDoc Imaging system (BioRad).
Fractionation and proteinase K treatment
250 OD yeast culture in exponential growth phase in SG medium supplemented with 0.67% casamino acids was collected by centrifugation (4000 g), washed in 20 ml H2O and resuspended in 12 ml of spheroplasting buffer (1.4 M sorbitol, 50 mM Tris-Cl pH 7.5, 40 mM 2-mercaptoethanol and 0.4 mg/ml zymolyase 20T) and incubated for 20 minutes at 30°C without shaking. Spheroplasts were centrifuged for 3 minutes at 1500 g at 4°C. The pellet was resuspended in 20 ml of cold lysis buffer (20 mM triethanolamine, 1 mM EDTA pH 7.2, 0.8 M sorbitol, 2 mM phenylmethylsulfonyl fluoride and 5 mg/ml each of leupeptin, chymostatin, aprotinin, pepstatin A and antipain). The spheroplasts were then lysed with a Dounce homogenizer (20 strokes). Lysates were cleared by centrifugation at 500 g for 5 minutes at 4°C and the supernatant centrifuged at 13,000 g for 10 minutes. The P13 fraction was then used for protease protection assays. P13 fractions were resuspended in lysis buffer and incubated with combinations of 0.3125 mg/ml proteinase K (Roche) and/or 5% Triton X-100 for 30 minutes at 30°C with gentle shaking. The reactions were then stopped with 10% TCA for 10 minutes at 4°C. Samples were then centrifuged at 13,000 g for 10 minutes at 4°C and the pellets suspended in 10 ?l protein sample buffer and separated by SDS-PAGE.
Oxygen consumption assays
Cells were grown aerobically at 28°C in the following medium: 0.175% yeast nitrogen base, 0.5% (NH4)2SO4, 0.1% KH2PO4, 0.2% DL-lactate (w/v), pH 5.5. Respiration assays of growing cells were performed in the growth medium. Samples of cells were harvested throughout the growth period, washed twice in distilled water and their dry-weight determined. Oxygen consumption was measured polarographically at 28°C using a Clark oxygen electrode in a 1-ml thermostatically controlled chamber. Respiratory rates (JO2) were determined from the slope of a plot of O2 concentration versus time and were expressed as natO/minute/mg dry weight.
For determination of cytochrome content, cells were harvested after 8 hours, washed twice with distilled water and concentrated to obtain 2 ml of a cell suspension of about 50 OD units at 600 nm. They were placed in a dual spectrophotometer (Aminco DW2000) and a differential spectrum (from 500 to 650 nm) was obtained from 1 ml of cells in the presence of 1 ?l of 70% H2O2 (w/v) (oxidized state) and 1 ml of cells in the presence of a few grains of dithionite (reduced state). Calculations of cytochrome c+c1 and cytochrome b contents were performed using an extinction coefficient of 18,000 M?1 cm?1 for the wavelength pairs 550-540 nm and 561–575 nm, respectively. The calculation of cytochrome a+a3 contents was performed using an extinction coefficient of 12,000 M?1 cm?1 for the 603–630 nm interval.
Explanation / Answer
Media used to grow yeast cell :
YPD medium : used for reuting yeast grwoth, contains yeast extract, peptone, dextrose/glucose.
YPG medium : Contains gycelore that dosenot grow to allow pet mutant.
YPAD medium : has adinine sulfat that is used to inhibit reversion of the ade 1 and ade 2 mutation.
Synthetical minimal medium : includes yeast nitrogen base without aminoacids.
Synthetical complete medium : has yeast nitrogen base with aminoacid supplements.
How to grow :
Yeasts are eukaryotic microorganisms whose genomes have been comprehensively studied and some have been sequenced.
They are relatively easy to grow under laboratory conditions.
Moreover, despite their small genome size, they display cellular features and processes that are highly conserved amongst most eukaryotes.
For instance, they have membrane-bound organelles, cytoskeleton, nuclear DNA, and transcription mechanisms that are similar to those found in higher eukaryotes.
Furthermore, yeasts have many well-characterized secretory proteins and pheromones. Several yeast genes involved in protease processing and secretion have also been identified.
Thus, yeasts can be used for several eukaryotic gene and protein studies with the aid of suitable molecular biology tools.
Some applications for yeast cultures include synthesis of protein expression systems, the study of specific gene or protein functions, and the analyses of novel protein interactions.
They are also used for many industrial applications such as fermentation, baking, and bioremediation.
Yeast culture media play a significant role in supporting growth in both small and large scale purposes.
Typically, a yeast culture medium includes peptone, yeast extract, and dextrose or glucose.
Even slight differences in media composition can yield yeasts with distinct growth characteristics
Test of yeast under differant condition :
Diffrent Temaratures :
Above ~95F (35C) - Yeast growth slows down and there is excessive production of bad flavors. Don't ferment in this range. Going significantly higher may kill yeast.
~77-95F (25-35C) - Yeast growth is fast, but there is more production of less desirable flavors (ammonia/sulfur as rumtscho notes, as well as odors that might be described as excessively "yeasty" or "beer-like"). This is mostly used when a fast fermentation is desirable or necessary, and it's best for enriched or flavored breads (with added sugar, oil/butter, eggs, other herbs/spices/etc.) which will mask the less desirable flavors. Sourdough ("natural" yeast) breads will often have even more substantial changes in flavor when fermented in this range, due to combinations of yeast and bacterial activity.
~68-77F (20-25C) - This is often considered the "optimal" fermentation range for bread. It's fast enough so you can bake in a reasonable time, but slow enough not to encourage bad flavors while allowing some enzyme activity to release more good flavors. This enzyme activity is particularly important for "lean" doughs (consisting only of flour, water, and salt), where all the flavor has to come from the flour.
~60-68F (15-20C) - This is the range sometimes advocated by artisan bakers for "slow" fermentation. The yeast develops more slowly, allowing more time for enzyme activity, as well as changes in dough structure. For sourdough breads, fermenting at a lower temperature can often lead for a more "sour" result, which some find desirable. The disadvantage is time.
below ~60F (0-15C) - These temperatures are generally used for "retarding" dough, rather than active fermentation. Again, they can be used to create more flavor or allow changes in structure and gluten formation. They can also be used for timing a bake while fermenting overnight or over a couple of days. Yeast will still grow somewhat down to refrigerator temperatures, but growth will be slower and slower.
UV :
Ultraviolet (UV) light is a type of radiation that generally has negative consequences on living cells. However, UV light has the opposite effect on yeast, as the rays can yield increased yeast productivity. The experiments into this interaction between UV light and yeast began.
Antifugal :
All anti-fungal drugs can have the tendency to give side-effects, ranging from the mild to the extreme.
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