Lab Three: Ethanol and Mitotic Effect on Yeast

What is the objective of part one of the lab?

Quantify the effect, if any, of environmental ethanol on mitotic activity in S. cerevisiae.

From an alpha-haploid yeast cell what is the first option in mitotic cycle?

To undergo budding

From an a-haploid yeast cell, what is the first option in. mitotic cycle?

To undergo budding.

The option other than budding for both a/alpha haploid cells is?

to undergo conjugation

What is conjugation?

Conjugation is a process where two yeast cells of opposite mating types fuse to form a diploid cell, allowing for genetic recombination and exchange of genetic material.

What is budding?

Budding is a form of asexual reproduction in yeast where a new cell grows off the parent cell, eventually detaching and becoming an independent organism.

When looking at the a/alpha diploid yeast cell there are two options to undergo mitosis. The first is budding, what is the second?

Formation of a spore

What is a spore?

A spore is a reproductive structure that is capable of developing into a new organism without fusion with another cell, commonly formed by certain fungi, including yeast. Spores are typically resistant to environmental stresses and can remain dormant until conditions are favorable for germination.

What does a spore do in yeast cells?

It reproduces into either an alpha or a-haploid cell.

What type of anaerobe is S. cerevisiae and what does that type do?

Saccharomyces cerevisiae is a facultative anaerobe, meaning it can survive in both aerobic and anaerobic environments. This organism ferments sugars into alcohol and carbon dioxide when oxygen is absent.

How much energy is formed during glycolysis?

During glycolysis, a net gain of 2 ATP molecules is formed from each glucose molecule.

If oxygen is present, then yeast shunts the other byproducts of glycolysis to other processes to form more ATP. What are those byproducts?

The byproducts are pyruvate and NADH, which enter aerobic respiration pathways like the Krebs cycle and oxidative phosphorylation to generate more ATP.

How much more ATP is formed in combination by pyruvate and NADH than the 2 net ATP from glycolysis?

A total of 30 to 32 ATP can be formed from one glucose molecule through aerobic respiration, including the contributions from pyruvate and NADH. Which when added to the 2 ATP from glycolysis results in 32 to 34 ATP in total.

Why do we replace NAD⁺ from another source other than glycolysis?

NAD+ is replenished through fermentation or the electron transport chain to ensure glycolysis can continue producing ATP anaerobically or aerobically, respectively. If NAD+ is not replenished, glycolysis would halt due to a lack of available oxidized coenzyme and the cell would die.

During fermentation what are the waste products developed?

The waste products of fermentation include ethanol and carbon dioxide, which are produced when pyruvate is converted under anaerobic conditions.

If oxygen concentration is low, then …

fermentation occurs in yeast, producing ethanol and carbon dioxide instead of going through aerobic respiration.

When we say ethanol fermentation does not capture anymore energy from glucose what do we mean?

There is a single purpose to ethanol fermentation, to replace NAD⁺ by converting glucose in to ethanol and carbon dioxide, allowing glycolysis to continue without further ATP production.

What is glucose first converted into during glycolysis?

2 pyruvate

What is the second product of glycolysis?

2 Acetyl-Coa which lead into Krebs Cycle

What is pyruvate converted into if it is undergoing ethanol fermentation?

2 Acetylaldehyde

What enzyme converts glucose to pyruvate?>

Glyceradehyde 3-phosphate dehydrogenase

What enzyme converts 2 acetylaldehyde into 2 ethanol?

Alcohol dehydrogenase

What is ethanol for the yeast cell?

A waste product and a metabolic toxin

What are we doing in today’s lab?

Assaying multiple concentrations of ehtanol on mitotic assays of yeast.

What is a live-cell exclusion assay?

A method used to evaluate cell viability by distinguishing between live and dead cells based on membrane integrity.

Why do we need to idetify dead cells?

If you include dead cells in the total population of cells the results may be skewed, leading to inaccurate measurements of cell viability and mitotic rate.

How do you differentiate between dead and living cells?

By using dye like trypan blue dye which does not enter the live cells but does permeate through the membrane of dead yeast cells. This allows visualization of live cells as unstained, while dead cells appear blue.

What is the first objective of the second part of the lab?

To learn how to construcct a hypothesis

What is the second objective of the second part of the lab?

To learn how to use the R programming environment to perform basic statisitcal analysis on experimental data.

What is the third objective of the second part of the lab?

To learn how to count cells using a hemocytometer

When performing an experiment…

you are testing a hypothesis

What will a good hypothesis do?

Give you a clearer picture as to what exactly the purpose of the experiment should be.

What is a null hypothesis?

A statement that proposes no significant difference or effect between variables, serving as a basis for statistical testing. (i.e. “treatment has no effect on viability”)

What is an alternative hypothesis?

A statement that suggests a significant difference or effect exists between variables, indicating that the treatment does affect viability. (i.e. “treatment has a significant effect on visibility”)

What is the minimum require control group?

A negative control

What should you set up the experiment with?

A control group and experimental group to compare results effectively.

What is the additional group included in the well-controlled experiment?

postive control

What is a positive control group?

A group in an experiment that receives a treatment known to produce a specific effect, ensuring that the experimental setup is capable of detecting changes.

What is the difference between the experimental and control groups?

The control group does not receive the experimental treatment, while the experimental group does, allowing for comparison of results.

Why should we replicate experiments?

In order to have multiple data points to analyze

If we discuss the results of observational and experimental results, what should we do?

Summarize the key findings and implications of both types of results to draw conclusions.

What is a statistical t-test?

A statistical test used to compare the means of two groups to determine if they are significantly different from each other.

What has to be done in order to accept/reject H₀?

A comparison of p-values against a predetermined significance level, typically 0.05, to determine if the null hypothesis should be accepted or rejected.

In a t-test what is alpha?

The threshold for significance, commonly set at 0.05, below which the null hypothesis is rejected. Also called the max acceptable probability.

in a t-test what is p?

The probability of obtaining test results at least as extreme as the observed results, assuming the null hypothesis is true.

During this lab what t-test is used?

The two-tailed t-test

What allows us to decide to accept or reject H₀?

comparison of the means using statistical methods

If P > alpha, then

the difference between means is not significant. we fail to reject the null hypothesis.

If P ≤ alpha, then

the difference between means is significant. we reject the null hypothesis.

During this lab we will utilize R-studio. What is an object like?

A noun

What is an element?

A single data point

What is a variable?

A named space that holds a data point

What is a vector?

A one-dimensional series of elements; each element has an assigned unique position within a vector.

What is the vector command of assigned an operator?

“< -”

What is the vector commands for mathematical operations?

“ + - x ÷ “

What does mean(ARG) do?

Returns the mean of the argument (ARG)

What does median(ARG) do?

Returns the median of the argument (ARG)

What doe sd(ARG) do?

Returns the standard deviation of the argument (ARG)

What does min(ARG) do?

Returns the minimum value of the argument (ARG)

What does max(ARG) do?

Returns the maximum value of the argument (ARG)

What does range(ARG) do?

Returns the difference between the maximum and minimum values of the argument (ARG)

In order to compare the samples what should each data set be converted into?

a vecotor

Step one of the statistical analysis is to determine if it is likely that the element of each vector were drawn from a normally distributed population. What is used to determine this?

The Shapiro test.

What is the command for the Shapiro test in R?

shapiro.test()

If the results of the shapiro test are that p ≤ 0.05 what must happen and what does ti mean?

The null hypothesis is rejected, indicating that the data is not normally distributed. Therefore, a non-parametric test should be used for further analysis.

If the results of the shapiro test are that p > 0.05 what must happen and what does ti mean?

The null hypothesis is not rejected, suggesting that the data is normally distributed. Parametric tests can be used for further analysis.

Step 2 of statistical analysis for this experiment is for if the data is not normally distributed. If thsi is the case what test will be used?

The Wilcoxon-Mann Whitney rank-sum test

What is the command for the Wilcox test?

The command for the Wilcox test in R is wilcox.test(), which is used to perform the Wilcoxon rank-sum test or the Wilcoxon signed-rank test.

What is the assumption that must be made for the shapiro test?

That the null hypothesis is that the values are consistent with normal distribution.

What is the assumption that must be made for the Wilcox test?

That there is not significant difference between the means

If the results of the Wilcox test are p ≤ 0.05, what does this mean?

It indicates that there is a statistically significant difference between the groups being compared. Therefore we reject the null hypothesis. meaning the data points come from different populations

If the results of the Wilcox test are p > 0.05, what does this mean?

It suggests that there is no statistically significant difference between the groups being compared, leading to a failure to reject the null hypothesis. Meaning that the data points come from the same population.

During Step 3 of the statistical analysis we compare the vectors to determine. whether their respective means indicate the vectors were drawn from the same or different population. To do this what test is used with the normally distributed data?

The t-test is used to compare means of normally distributed data.

What is the R-studio command for the student t.test?

The command is t.test(), which is used to perform a t-test in R.

What is the assumption made for the student t.test in this experiment?

The null hypothesis states that there is no difference between populations.

If the results of the t.test are p > 0.05, what does this mean?

This means that there is not enough statistical evidence to reject the null hypothesis, indicating no significant difference between the group means, and they are from the same population.

If the results of the t.test are p ≤ 0.05, what does this mean?

This indicates that there is sufficient statistical evidence to reject the null hypothesis, suggesting a significant difference between the group means, and they are likely from different populations.

Why is ethanol fermentation important?

It refuels glycolysis when oxygen is low and recycles NAD⁺ to NADH

Is ethanol toxic to yeast?

yes

Does ethanol inhibit cell budding or increase cellular death?

Ethanol can inhibit cell budding and increase cellular death in yeast, affecting their growth and reproduction.

What is special about the scientific process?

It is an ongoing process.

What is used to test hypothesis?

Experiments and observations are used to test hypotheses in scientific research.

What is the p formula for H₀?

p > 0.05

What is the p formula for H_A?

p ≤ 0.05

What is p?

The probability of getting a certain result o this time.

What is step 1 of the lab?

Centrifuge the prepared cells and discard supernatant to isolate the yeast cells.

What is step 2 of the lab?

Resuspend the yeast cells in PBS and add trypan blue to the mix. Incubate at 25ºC for 3 minutes

What is step 3 of the lab?

Add to a hemocytometer and analyze the results.