Saccharomyces cerevisiae - Science topic

I haven't done it, but I found this publication by J. P. Aris where they describe a method to isolate yeast nuclei:

yes sure, but in this case you should not continue to call it BY4743

Hi there,

These are strong constitutive promoters and terminators. Any combination should be OK... I have successfully expressed simultaneously 6 human proteins from genomic insertions using the following combinations: ProPGK1+terTDH1; ProTDH3+TerADH1; ProHHF2+TerSSA1; ProCCW12+TerENO1; ProTEF1+TerENO2; ProTEF2+TerPGK1. The most crucial point being to optimize sequences for expression in yeast.

Disaggregating yeast chains can be challenging, especially when working with certain species like Saccharomyces cerevisiae that tend to form large cell aggregates. Here are some techniques you can try to improve yeast cell separation:

To address this problem, you can use these strategies, as I mentioned below:

Remember to perform thorough optimization and validation of your experiments to ensure the reliability of your results.

Thank You!

Do you mean you do not have access to get it? If so, see the attached.

Hi Timothy K. Craig

There could be several reasons why you're not achieving the same transformation efficiency as the paper. Here are a few things to consider: Quality of Plasmid DNA: Ensure that your plasmid DNA is of high quality, pure and not degraded. Use a reliable method to isolate and purify your DNA. Competency of Yeast Cells: The competency of yeast cells is critical. Make sure that the cells are appropriately prepared, not too old or too young, and handled gently during the preparation process. Plasmid DNA Concentration: The amount of DNA used can impact transformation efficiency. Verify the concentration of your plasmid DNA using a reliable method such as spectrophotometry or Qubit fluorometer. Transformation Protocol: Be sure you're following the protocol closely. Small details, like the temperature and timing of heat shocks or the voltage and cuvette gap in electroporation, can make a big difference. Media and Growth Conditions: The choice of media, pH, temperature, and even the type of agar can affect transformation efficiency. Strain Variation: Different yeast strains can have different transformation efficiencies, even with the same plasmid. Make sure your yeast strain is identical to that used in the paper. Experiment Reproducibility: Even when a protocol is followed precisely, achieving the exact same results can be challenging due to variability in conditions and materials.

Thank you!

You can always use recombineering using a Lambda red/ICP8 based system.

Hi there,

Integration occurs via homologous recombination in yeast. To optimize this integration you have to linearize the plasmid within the region where recombination events are expected so that extremities of your linearized plasmid are homologous to the site of insertion. The plasmid has to bear a selection marker (as linearized plasmid is not stable in the cell, selection will be made on insertion).

Saccharomyces cerevisiae amount 1% or 10% w/wbiomass ???

Dear Diana Villarreal-Huerta

We also experienced a similar issue. When RNA is isolated from stationary phase culture, the quality is poorer (according to RQN or RIN value) than RNA isolated from log phase culture. We used commercial kit RNeasy Plus Mini Kit from Qiagen. The obtained RIN values for all stationary phase RNA samples were above 7.5 which is acceptable for the high throughput sequencing, whereas the RIN/RQN values of log phase RNA samples were more than 8.5. We also observed that following purification of mRNA, incubation at room temperature leads to RNA degradation proportionally. I would like to encourage you to use commercial RNA isolation kit and must use DNase treatment following RNA isolation to increase yield and quality of RNA samples for high throughput experimentation.

Best wishes.

Combining feed enzymes, minerals, and probiotic microbes can have positive effects on the growth and health of aquatic animals, as each component plays a different role in supporting their nutrition and gut health.

Probiotic microbes are live microorganisms that, when ingested in adequate amounts, provide a health benefit to the host. They help to improve gut health, nutrient utilization, and disease resistance. Feed enzymes, on the other hand, are proteins that break down complex nutrients in feed into simpler forms that can be more easily digested and absorbed by the animal. Minerals are essential nutrients that play important roles in various physiological processes, such as bone formation, enzyme activation, and nerve function.

When combining these three components, it is important to consider the specific needs and characteristics of the aquatic animal species, as well as the production environment and feeding conditions. In general, a balanced composition of feed enzymes, minerals, and probiotic microbes can be achieved through a combination of the following steps:

Overall, combining feed enzymes, minerals, and probiotic microbes can be an effective way to improve the efficiency of aquatic animal production, but it requires careful consideration of the specific needs and characteristics of the target species and the production environment.

I am currently experiencing very similar problems. Did you find any solution or more information on how the blast search works?

Hi there,

These small size RNA are mainly tRNAs and probably 5S RNAs too. If you want to get rid of them you should use IEC (small RNA being eluted at lower ionic strength than rRNAs). Nucleobond RNA/DNA kit is what I'm using to separate differents RNAs.

Thank you Amy Klocko , Hanna Alalam , Francisco Javier Alvarez and Zaleha Shafiei for writing back.

Hi!

1) Try to check your protocol by calculate CFU on the solid media (Saburo for Candida); 2) to minimize time between preparation inoculum and starting the procedure cells calculation (this is important for escape inoculum effect); 3) try to use more QC on every steps.

BW

Dear Partha Dey

It would be easier to understand your trouble if you mention which type of protocol you are following and which type of DNA (plasmid or DNA cassette, its size and amount), selection marker (antibiotic or auxotrophic) etc. with your question.

The problem is whether you have a selection for the plasmid. The normal lab strains of S. cerevisae are often auxotrophs that are used for plasmid selection but a wild type strain would not have these markers to use. Note that bakers yeast is also still S. cerevisiae but just a notably different strain than the domesticated lab versions.

During DAPI staining of yeast cells, the cell wall of yeast cells also get stained. I have also used 0.1% Triton X. But still getting the same problem.I am using 1μg/ml DAPI. I also have increased the washing. Please suggest any solution for this.

When I select for Hygro or G418 resistance I plate my transformed cells on YPD first and incubate o/n then transfer with velvet on drug-containing plate. It helps to the cassette to be expressed correctly. In my hands it helps a lot.

Best

Thank you Francisco Javier Alvarez

Xiao Yi NLSs are known to be involved in the active transport of foreign proteins and probes into the nucleus. However, the stimulation of plasmid DNA nuclear localization has yet to be fully validated.

How do you get such high transformation efficiency of Saccharomyces cerevisiae? Can you share your experience?

Sure Azka Saleem

Steps u can consider following.

1. Download the SGD data as a fasta file/if separated as many files, u can merge all into single fasta file.

2. Make a list of IDs in MS excel whose sequences you need to extract.

3. Install TB tool in your dekstop, and search for fasta extract option, where u need to provide the database fasta file and the IDs and click ok.

4. In the output file you will have your required IDs sequence data.

If you still find it difficult to perform, u can mail me in nkrdas2@gmail.com

Thank you for your answer sir

Saccharomyces cerevisiae is not like the molds, it doesn't produce spores that would travel through the air. I worked years ago in the same biosafety cabinet with yeast cells (of Candida albicans, which is pretty similar to S. cerevisiae) and never had contamination problems. Plus, you can clean the biosafety cabinet and use the UV light in between experiments. So, I would not worry about that.

Fundamental misunderstanding, I'm afraid. Though it's great that you're thinking these things rather than just assuming you know what's going on.

DNA is always transcribed in the same direction, it's just that DNA has two strands, which run in opposite directions.

There is no difference between a gene going 5'-3' on one strand, and a gene going 5'-3' on the other strand, even though those two strands are wound together such that each runs in the opposite direction to the other.

A gene can be encoded on either strand of the DNA, as here. The RNA polymerase copies from one strand, and the other strand in that region is ignored. RNA polymerases only 'see' one strand at a time, and that's the strand they bind to

(slightly confusingly, they bind to the non-coding strand, and they travel in a 3'-5' direction, because they are producing a complementary sequence, i.e. a copy of the coding strand, which consequently runs 5'-3').

The resultant mRNA is single stranded, is a copy of the coding strand sequence (with U in place of T, obviously) and runs 5'-3'.

Note that genes rarely overlap between strands (i.e., HTZ doesn't overlap with PDB3) because this would require a region of shared anti-parallel coding sequence (which is both much more vulnerable to mutational change, and a lot more challenging to evolve). Some viruses do in fact have multiple reading frames that overlap on antiparallel strands, because viruses are awesome, but this is because viruses are under huge selective pressure to have small genomes, while higher eukaryotes have essentially zero pressure for this (the human genome is like 90% filler sequence, for example).

Cual es el proposito de su investigacion?

Si es su trabajo con la cepa, deberia repetir el experimento evitando la contaminacion de muestras

to be 100% sure that there is no carbohydrate in the medium I would NOT USE YPD. There might be something (although in low amounts) in peptone and yeast extract. WI do recommend to use chemically defined minimal medium, such as Yeast Nitrogen Base, which is available from various suppliers.

Hi Daniel,

I would highly recommend you not to follow that paper because the authors would not provide *any* help, feedback if you face any problem.

For chromosome homology, 1000bp that was used in that paper is absolutely overkill. Don't bother yourself. Even 300-400 bp homology works well.

If you want a clear transformation protocol let me know and I will send you. However, I would strongly suggest to use this protocol:

Hi Pietro,

Without a microscope or specific media culture is hard to know what is your contamination. It could be another yeast or a bacteria.

As Dominique Liger said, it would be easier to restart from your yeast stock. Make sure your new culture media is sterilized!

Your right of course, we used to use a Pep4 ko for protein expression. We also knocked out Kex1 and kex2 when I worked in industry, interesting you have a multi protease gene knockout and it still has normal functionality. Thanks for sharing

Try adding in some protease inhibitor in case your sample is getting degraded. If GFP is being cleaved, that will show up as a GFP positive band (about 26 KD for denatured). Also, sometimes antibodies do degrade/expire. Is anyone else using that antibody and are they also having issues with lack of bands?

Hellie Gonu dear, its not only yeast that can effect the production of ethanol. temperature, media ingredients and concentration can also play a vital role in it. what temperature are you using in fermentation?

Hi,

I have 2 points here and i hope they help.

1. I am assuming that you are using a commercial master mix for PCR. for low GC content template, you might need to have more Mg2+ in your PCR reaction. Maybe you want to try to supplement some MgCl2 in your PCR mix. (I had the problem when I PCR low GC products from Candida albicans genome, and supplementing Mg2+ helped.)

2. Have you checked the primer dimers across the pairs of primers you have? Could it be that some of the primers form strong dimers which resulted in missing of PCR products?

Séchage à letuve

Hello Danielle Nel;

Regarding your question, congratulations for the detail that you gave to explain your experimental procedure. This is good so that one can give you a more accurate answer or advice.

As other collegues have mentioned, the long time you leave the batch reactor running could be the cause to lower ethanol productivity at the end of the fed-batch reactor. Your guess seems to be right regarding the growth phase at which you started the fed-batch stage; in that case you could have started in the stationary or death phase.

I suggest that you first characterize the cell growth in your batch reactor. It is recommendable to start the fed-batch in an active growth-stage of your batch (full exponential phase), prior to the stationary phase. Inicial cell concentration of the fed-batch stage may be also important for good yields, so that is why commonly it is used the late exponential growth-phase for fed-batch start. After you have this information, you can define the time at which you would start your fed-batch stage. An obligued reference for this is the classical text book by Bailey and Ollis, Biochemical Engineering Fundamentals.

Apart from this, you said that you are comparing your results to a report of the literature. In this regard, you have to take into consideration the biochemical characterization of the SSL used in both studies, as well as the yeast strains used for fermentation. These are both important parameters that would influence the yields of the reactor. As SSL is a biological waste stream, it can vary widely in its chemical composition even from batch to batch.

I hope this helps for your research.

Best regards.

If your data are gaussian, you may try Pearson correlation. cor.test() in R. Run a Shapiro-wilk test first to test for normal distribution and then test if the covariance is linear.

First of all there are inducible promoter systems in yeast already so I'm not sure why you would not use a native one (ADH, galactose, many others).

It is possible to use lacI repressor to act as an inducible repressor in eukaryotic systems, that has been done because you merely need to engineer the operator binding site into a region of the promoter where repressor binding would interfere. IPTG induction will work in any system. However it is not that broadly used, but the tetracycline inducible system is fairly commonly used.

Arabinose is more problematic because in E. coli the arabinose system is a positively regulated system where AraC protein acts as a positive effector of transcription. Eukaryote and prokaryote polymerases are too different to expect the Arabinose system to work as a positive effector so I would not expect it to work unless you were only going to use it as a repressor like with LacI.

While you can use point mutations to introduce premature stop codons close to the start of the open reading frame, this would leave room for revertants to occur. As such, deletion of the genes in question is safer. See

Check https://scholar.google.ch/scholar?as_ylo=2020&q=yeast+gene+disruption&hl=en&as_sdt=0,5&as_vis=1 for more recent examples

Hello Kedric Milholland

May be you should try other methods of total protein quantification of your lysate. Usually, for Western Blot, Bradford or Bicinchoninic acid (BCA) assay is used for quantification of protein from the lysate. Using standard solutions of bovine serum albumin (BSA) you can produce a calibration curve of absorbance versus mass concentration. Assuming the analyte-proteins react in the same manner as the BSA standard, the unknown concentration can be determined. Then you can load 10ug - 20 ug of the total protein on the gel.

Best.

Hi Dorine

please take a look at this paper for LncRNA discovery in yeast:

all the best

fred

Thanks you. I`ll check out if I`ll have anything looking like a Kozak sequence in the plasmids.

Hi there,

y

Your strain seems to be deficient for cell arrest induced by alpha factor (deficient for some STE proteins of the response pathway?). What is its genotype? You get a partial efficiency of the pheromone. You might try to introduce bar1 deletion in your strain in order to improve cell sensitivity to alpha factor. Otherwise you may try other block and release strategies (hydroxyurea in S phase or nocodazole at G2/M).

Hello,

Getting an exact sequence for this plasmid would be a difficult since at least two parts of it where constructed by BAL digestion and the authors of the original paper that constructed the plasmid did not describe in details. However they indicate that the majority of the backbone comes from YEP24 (not the gal1 promoter) and a schematic diagram of YEpTOP2GAL1 can be found here:

I suggest you compare the schematic with the YEP24 map from this link:

take the common sites and sequence from there (for example the URA3 marker is closest to the TOP2 insert and hence you can design accordingly). A couple of good sequencing reaction will help you in inferring the entire sequencing of the plasmid.

I am coming back with some updates on the questions i asked before.

1. I tried transforming 14kb plasmid with ura marker into yeast; it worked perfectly well and i had several clones on the selection plate.

2 . For second plasmid tarnsformation, I prepared new competent cells containing 8kb plasmid with trp marker and transformed 14kb plasmid with ura marker. I increased the incubation time to 2hrs. i had colonies on plate but the efficiency is too low as compared to single plasmid transformation. Nevertheless, few colonies served my purpose.

Hi Joshua,

the analysis does not have to be done with PD, it was recommended this was used. PD is an expensive commercial product, but is a very capable piece of software. MaxQuant is free and an excellent package fully capable of performing the analysis required in this instance. MaxQuant is also capable of working with PTMs, so yes, I'd start there. Good luck.

Hi Gayatri,

If you mean an online platform like those ones available for bioinformatics, not unfortunately. There are however a bunch of amazing videos available on YouTube, and. I do believe you can always ask your yeast questions here on researchgete, and there are many to help you (including me).

Kindly check the following links:

I have nothing to add to the comments above, which are right. You really need a positive control to show that both the transformation and the media are correct.

(there will be some regulation,but it will be very difficult to control. however, you cannot avoid any regulation from the start). Sorry I have taken all this time to answer

6/18/21

Dear Adam,

I'll try to answer your questions.

I wouldn't use TCA (trichloroacetic acid) to ppt your enzyme. TCA is a very strong acid and is sometimes used to stop enzymatic reactions. Although it may ppt your enzyme, it will denature it. Acetone has been used to ppt and recover enzymes with activity. However, since it is an organic solvent, it, too, can sometimes denature enzymes. It just depends on the "robustness" of the enzyme Some enzymes can tolerate it, some cannot..

Your best option is ammonium sulfate (AS). This has long been used as a "safe" protein/enzyme precipitant. Not only can it ppt enzymes, but the enzyme ppt is often stabilized by the AS. Keep in mind that as a protein precipitant, AS is non-specific, that is, it will precipitate many other proteins from your culture fluid besides your endoglucanase, so your precipitated enzyme will not be pure, but a mixture of many proteins. AS is usually employed as as a first step in purification of proteins. The protein ppt (containing many proteins) is then subjected to other downstream purification techniques such as ion exchange and/or size-exclusion chromatography to recover the protein (enzyme) of interest in pure form.

If you don't have the resources to do the downstream purification techniques, you can do AS ppt'n in stages. Different proteins can be ppt'd with different concentrations of AS allowing you to effect a partial purification of the protein of interest. E.g., You can add AS to your culture medium to reach an AS concentration of ~33% saturation. (Do the precipitation in the cold.....cold room or ice bath. Do not freeze.) Allow 1-2 hrs for the protein to ppt (overnight is OK.). After this, spin down and recover the ppt'd protein. Recover the supernatant and add more AS to reach a concentration of 66% saturation and repeat the above to recover a 66% AS protein ppt. Recover the 2nd supernatant and add more AS to reach 100% saturation and repeat the above. You will have AS ppts from 33-, 66- and 100% saturation. Test the AS ppts for enzyme activity. If you are lucky, most of your endoglucanase will be concentrated in one of the 3 ppts; most of the other (unwanted) proteins will (hopefully) be in the other 2 ppts.

If you need to remove the AS from the recovered enzyme, dialysis in the cold against a suitable buffer will work. Be advised that the AS in your preparation will "draw" liquid from the dialysis buffer into the dialysis bag and cause the volume to increase, so be sure to leave sufficient "headspace" in the dialysis bag.

I hope this information helps you.

Bill Colonna Ames, IA USA williamjcolonna@gmail.com

We are working in the field of Mycology since 1973.We always used chloramphenicol in the mycological media, such as Sabouraud dextrose agar and Pal sunflower seed medium to inhibit the growth of bacteria.We did not use ampicillin/kanamycin in YEPD.I do not think there will be any problem to grow/maintain Saccharomyces cerevisiae in YEPD supplemented with ampicillin/kanamycin.

Check https://link.springer.com/article/10.1186/s13068-016-0554-6

Hi there,

There is no theoritical value as there are too many parameters which can't be easily controlled: for rich media for instance, the origin of YE and peptone will have an impact as these are complex media. Also the aeration, the flask volume will affect the growth rate. Also the strain (even if described as wt) will have an effect. So just run the experiment in your own condition! As an indication wt strain has a generation time of 90 to 120 minutes in YEPD... If your intention is to compare strains, make sure to run all the sets of cultures in parallel using the same batch of media. As mentioned above, experiments in SC are more easily reproducible as the composition of the media is perfectly controlled.

20 ml is more than enough for intracellular proteins, for secreted ones I guess very much depends on the nature of the protein. You van find detailed protocols for TCA extraction in the web (just google TCA protocol for yeast). I attach my personal one. Good luck

M

Saccharomyces cerevisiae (bread yeast) produces ascospores. It belongs to the class Hemiascomycetes which has no ascocarps.

Dear Peng Zhan,

I have attached one article which the metabolic pathway of Saccharomyces cerevisiae has been shown and explained. I hope it would be useful.

I think the only difference between pRS316 and pRS416 is that some restriction sites were modified so that they would be unique in pRS416. Some of the sites in the multicloning site were not unique in the 3 series and existed elsewhere on the plasmid.

Hi there,

The following should help:

Hi there,

Did you check total on total protein extract? SDS/PAGE is not enough to state if there is expression or not (low level of expression will not be noticed if the POI migrates the same as an intense band of endogenous proteins). The difference between the 2 situations is of importance: if no expression it means there is either something wrong with the construct or the strategy for expression. If little level can be detected it might indicate the protein is not stable and degraded at a rate similar to the synthesis or if insoluble it might tell there is an issue with the protein folding (galactose induction is a strong induction and may lead to misfolding/mistrafficking). Have you got an other way to detect the protein like WB?

And https://www.sciencedirect.com/science/article/pii/S2405844020312366

Dear Amaldo Firjarahadi Tane thank you for your interesting technical question. In addition to the valuable answer and link provided by Maurice Ekpenyong please also have a look at the following potentially useful articles entitled

Determination of the molecular weight of Saccharomyces cerevisiae nuclear DNA

and

A re-evaluation of the molecular weight of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase

Also check https://www.hindawi.com/journals/isrn/2014/532852/

Saccharomyces bayanus is a species. There is a variant of it that is S. bayanus var. bayanus.

I use the Mycobank.org database to check for species consistency...it generally has all the possible pseudonyms.

https://blast.ncbi.nlm.nih.gov/Blast.cgi

Worked in the past with different S. Cerevisiae strains ( some were high flocculation) and never saw what you are describing. I think J. Stolz is right suggesting a contamination (some bacteria would make a though film a the top of a liquid culture e.g. acetobacter or kombucha SCOBY). Some strains of S. Cerevisiae could definitely clump in the top foam but not the point of having something difficult to re-suspend. Eventually if your shaker stopped functioning for a long time during the cell growth that could happen. I would also advice to have a look under microscope.

I used the same method to identify mutants defective in surface Fe3+ reduction so it might be that that is what you see and not respiration. As controls I always included a heat-inactivated (not too hot or your agar is melting, rather incubate longer) or UV inactivated plate, where cfus remain white. Also, antimycin A works as an respiration inhibitor. So if you still get a signal with cfus grown on that it's probably Fe3+ reduction you see...

Quality requirements vary depending on the downstream application. RNA sequencing and microarray experiments require high quality RNA samples with specific purity and integrity (A260/A280 bigger than 1.7 and RNA integrity number (RIN) bigger than 8.0). In contrast, for qPCRbased assays and northern blot analysis, samples with lower quality scores may be used. I advise you to re-isolate your RNA samples. If the 260/280 ratio is lower than 1.7 this means there are impurities on your RNA sample (like residual phenol, guanidine and proteins). For more information, you can read this article

Hello Michele

Please consider the following points:

1. First and foremost, the amount of protein loaded per lane is too high. You should load about 15ug to 30ug protein per well. The gel can hold more of the protein than the membrane binding capacity. Therefore, the excess protein may stay in the gel.

2. Higher percentage gel can slow the transfer process. So you need to increase the transfer time and if required even the voltage. Since you have both HMW and LMW proteins you need to optimize the transfer conditions.

3.Methanol is a fixative generally used to improve the adhesion of smaller proteins to the membrane. Since you have more of HMW proteins you need to lower the percentage of methanol in the transfer buffer. Also the binding of protein to the gel will be reduced.

Good luck.