Overview

While working on this project you will:

• Get to know the teaching staff and other students, and get used to working in the lab
• Discover concepts around the scientific method and experimental design
• Learn about note keeping and practise writing your lab book
• Write a basic report

Timetable

Introduction to the scientific method

A first session will introduce you to some important concepts regarding the correct conduct of scientific research.

Project introduction and planning

The following topics will be covered in the session on [day]. The text below is for your future reference (but of course, feel free to read it over before the session if you like).

Theoretical introduction

In this experiment we’ll be using budding yeast, Saccharomyces cerevisiae. This single-celled eukaryotic microorganism is used for a number of day-to-day applications such as baking and brewing, and is a widely-used model organism. It is very useful because it can be grown quickly and cheaply in the lab using liquid or solid media; has a small, easily manipulated genome; and shares many basic cellular functions with higher eukaryotes such as humans.

Respiration

You will be working with other students from your tutor group to investigate the ideal conditions for S. cerevisiae metabolism. Specifically, you will be investigating its respiration – the metabolic process of breaking down sugars, generating adenosine triphosphate (ATP) and other energy-carrying molecules which can be used to fuel the other processes that the cell needs to perform to live. This will be studied in much more detail in your [Biochemistry] module and I will only provide some basic information here to help you understand our experiment.

During respiration, glucose sugar is broken down to pyruvate during a pathway known as glycolysis (two pyruvate molecules and two ATP molecules generated for each glucose molecule that enters the glycolytic pathway).

There are two pathways for the processing of pyruvate: 1) aerobic respiration (requires oxygen), which is more efficient and the preferred pathway in nature, and 2) anaerobic respiration (also known as fermentation), which is useful for some industrial processes because it generates ethanol or lactate as a by-product.

In aerobic conditions, pyruvate is fed into the citric acid cycle: this leads to the generation of a small amount of ATP, as well as electrons that can drive oxidative phosphorylation in the mitochondria. This is where most of the energy in aerobic respiration is generated.

Importantly for our purpose, this process also generates CO₂. This is what we are going to be measuring in our investigation.

Metabolism of diverse sugars

Glucose (containing six carbons) is the “ideal” sugar that enters into this pathway. However, a number of other sugars are also available to the cell.

Fructose and galactose are alternative six-carbon sugars, whereas molecules such as sucrose, lactose, and maltose are essentially made up of two of these joined together (twelve carbons in all). These sugars, and how they can be used by human and/or yeast cells, are illustrated in [the figure on the next page].

Our experiment

We’re going to use the “yeast race” method as a way of measuring respiration in different conditions. This method involves incubating a yeast culture in a narrow tube. As respiration takes place, the carbon dioxide generated creates bubbles in the culture, and the resulting foam rises up. The level of the foam can be measured and is proportional to the amount of respiration that occurred.

Basic procedure

1. Add reagents to a 50 ml Falcon tube in the following order (this is important to make sure everything goes into solution):

1. 12.5 ml distilled water (in the 37^oC water bath): it’s accurate enough for our purpose today to measure this simply by pouring water up to the line on the tube
2. Any sugar that you choose to add. MIX WELL by shaking the tube with the lid on, and ensure all of the sugar is in solution
3. 4 g plain white flour
4. 0.5 g baker’s yeast. MIX AGAIN by inverting the tube gently (with the lid on) and ensure everything is in solution

2. Remove the tube lid and note the level of your solution (should be around 15 ml).

3. Incubate at room temperature for 60 min. Record the level of the bubbles every 15 minutes: record the volume using the lines on the side of the tube.

Our experiment

You may use up to 12 tubes.

For each reaction, you need warm distilled water, plain white flour, and baker’s yeast, as above.

Other reagents you have at your disposal: glucose, sucrose, lactose, and fructose. Maximum amount of sugar to add per tube = 1.5 M.

Experimental planning task

You will be working in groups for the planning and for the execution of this experiment – each tutor group will split into two teams (about 4 people per group).

Questions to guide your planning – in your group, work through these in order:

1. What is your hypothesis?- Together, decide on a specific, testable statement. What would you like to know about yeast respiration? You might be interested in the effect of sugar concentration or the efficiency of different types of sugar. Make sure you don’t try and do too much all in one go! It’s more important that you have a clear question with appropriate controls than to investigate everything at once.
2. How to set up a reliable experiment? Think about negative and positive controls, the fair test, and duplication. [Positive control: 0.5 M glucose should give you a good amount of foam].
3. Variables. Which extra reagent(s) will you add to each tube? Use the table below to make sure you are adding comparable amounts of different sugars.
4. Write everything out clearly so you can remember the plan tomorrow!
5. Ask a demonstrator to check your work before you leave. If you haven’t finished by the end of the hour, that’s OK – we can check it at the beginning of the session tomorrow.

Molarities for the sugars we’re using

When planning our experiment, it’s important to use the same MOLARITY rather than the same MASS of the different sugars. This means that we are adding the same number of sugar molecules into the reaction mixture (even though the molecules are of different weights). To save time today we are providing the following conversion table.

Of course you can use different concentrations within this range if you like – you are not limited to these numbers!

Practical work

• The session will begin with some advice on writing your lab book.
• Then, you will work in your groups to carry out the experiment you planned.
• Record your protocol and your results in your lab book

Analysis and write-up

• You should arrange to bring at least one laptop per group to the session (if this is going to be a problem, let us know). You will create a graph, analyse your data, and then complete the mini-report using the template provided.
• The report template is on the following page.
• Guidance will be provided on how to draw helpful graphs using Excel / Google sheets
• Sections of the report:
  • What we asked (Hypothesis): 1 sentence
  • What we did (Materials and Method): 3 sentences
  • What we saw (Results): Figure and description : 5 sentences
  • What we found out (Discussion): response to the hypothesis: 2 sentences
• If you had trouble during lab session 1 and couldn’t gather any data we can consider doing a repeat (or perhaps a smaller experiment) today – come and see a member of staff and we can decide what to do.
• When you have finished, [describe plan for sharing with tutor]. You will discuss your experiments in the next group tutorial.

Group tutorial: informal discussion of results

An important part of being a scientist is chatting about your data with other researchers. Therefore, in your next group tutorial there will be an opportunity for you to share your experiment with your tutor.