Yeast Mutagenesis

Author(s): Kamena Kostova, Darienne Myers, Larry Cohbra

Lesson Overview

Grade level(s):

High School (9-12), Grade 9, Grade 10, Grade 11, Grade 12

Subjects(s):

Biology/Life Science

Topic:

Cellular biology, Cancer

Big ideas(s):

Mutagens induce mutations in the genetic material of cells. Harmful mutations can result in genetic disease while other mutations may be neutral or beneficial to an organism.

Vocabulary words:

mutagen, mutagenesis, model organism, cancer

What you need:

- Access to a wet laboratory, ideally a yeast lab for supplies and shakers.
- Yeast strain (UV-sensitive) (obtain from Carolina Biological Supply, or other)
- SD ­Ura media (synthetically defined yeast media without Ura) to maintain yeast culture (~50 ml).
- YPD plates (buy online at Carolina or other supplier, or pour your own)
­- 5-FOA plates (Pour your own or obtain from local university lab)
- Sterile container with lid
- Sterile pipet to measure 100 µl
- Pipet bulb or pipet pump to use with the pipet, if needed
- Sterile test tubes with caps (optional)
- Sterile distilled water (optional)
- 70% or 95% ethyl or isopropyl (“rubbing”) alcohol
- Waterproof marking pens with fine tips ­ 1/student pair
- Sterile, flat toothpicks
- Aluminum foil
- Paper towels
- 22 nm filters for filtering the mutagens
- “Mutagens” ­ tea, coffee, soap, dishwashing detergent, deodorant, parsley, diet coke, etc.
- Plastic disposable pipets
- Place to incubate the yeast (30 °C incubator or room temperature)
- Sterile Polystyrene Petri dishes (100 x 15 mm)
- Bunsen burner (optional)
- Eppendorf tubes
- Microscopes
- Microscope slides
- Iodine to stain yeast
- Cotton swabs / Q tips (for doing cheek swabs)

Grouping:

Students work in pairs or in groups of 3's.

Setting:

Laboratory/classroom for the investigation.

Time needed:

Five lab periods (60-80 minutes each) and an additional period for student presentations. Allow at least a few days (3-4 days minimum) to grow the yeast on the plate after Day 2 and Day 4 of the investigation.

Author Name(s): 
Kamena Kostova, Darienne Myers, Larry Cohbra
Summary: 

In this laboratory investigation, students learn the concept of mutagenesis and explore how different substances can act as mutagens. The experiment utilizes a strain of yeast that lacks several DNA repair mechanisms, allowing it to accumulate mutations after exposure to mutagens. Students expose this strain of yeast to everyday substances (soda, soap, Aspirin, glue, etc.) and record the effects. Using this data, students will infer the affect of these substances on living organisms. 

Prerequisites for students: 

Basic microscopy

Lab safety

Learning goals/objectives for students: 

Students will be introduced to the use of a simple model organism (yeast) to gain insight on basic biochemical processes (such as DNA repair) in more complex organisms.

Students will understand the concept of mutagenesis and how it relates to genetic diversity and diseases such as cancer.

Students will understand the purpose of statistical analysis when interpreting data.

Students will formulate a hypothesis, plan an investigation, analyze the data and complete a scientific presentation.

Content background for instructor: 

This lesson is spread over 6 class periods:

  1. Introduction to cancer and mutagenesis
  2. Mutagenesis lab
  3. Data analysis from mutagenesis lab
  4. Modified mutagenesis lab
  5. Data analysis
  6. Student presentations 

Researchers use a model organism to directly test their hypotheses regarding biological processes. The knowledge gained from these studies can often be applied to more complex organisms, including humans. Commonly used models include the bacterium Escherichia coli, the fruit fly Drosophila melanogaster, the zebrafish Danio rerio, the nematode Caenorhabditis elegans, the mouse mus musculus and the yeast Saccharomyces cerevisiae among many others. They often share characteristics such as a sequenced genome, a short lifespan, ease of care and genetic manipulation.

Different model organisms can be used to answer different questions. For example, while a mouse model is useful in studying the development and progression of various cancers, basic cellular processes can be easily studied in smaller organisms such as yeast.

Yeasts are unicellular fungi used extensively in biomedical research as model systems to study fundamental cellular processes. For example, they have been used extensively to better understand genetic and biochemical processes in living cells such as cell division, metabolism, DNA replication, recombination and repair, gene function and regulation etc.

The most commonly used strain in research is Saccharomyces cerevisiae, also known as Baker’s Yeast (or Brewer’s Yeast) for its uses in baking bread, brewing beer and fermenting foods. S. cerevisiae are round and are typically 5-10 µm in diameter. They reproduce most often asexually by budding but can also reproduce sexually through the formation of haploid cells (meiosis) with two mating types.

Mutagenesis refers to the induction of genetic mutations (alterations in genetic material in a cell) due to exposure to biological, physical or chemical agents called mutagens. Most somatic (non heritable) mutations occur spontaneously during DNA replication and are repaired by the cell DNA repair mechanism. Mutagens increase the rate of mutations that occur, increasing the possibility that the mutation becomes stable. Examples of physical mutagens include ionizing radiation (X-rays), and UV light. Biological agents also exist: viruses can induce mutations in a cell when viral DNA is inserted into the genome. Additionally, many common household items have mutagenic properties, such as bleach, insecticides, and some dyes. Toxic compounds including benzene, asbestos and nicotine are often mutagenic as well.

Mutations that alter the function and/or regulation of a gene can cause disease. For example, mutations in genes that regulate the cell cycle can lead to unregulated and proliferative cell division that may turn into cancer. Carcinogens are therefore mutagens that can lead to cancer development in organisms.

Types of mutations

Big genetic alterations can result in frame shift mutations in protein coding DNA, where the gene can no longer be “read” properly.

  • Insertions: one or more nucleotide base pairs is inserted into a DNA sequence
  • Deletions: one or more nucleotide base pairs is lost from a DNA sequence
  • Inversions: A fragment of a chromosome is reversed and re-inserted.
  • Duplications: duplication of a region of DNA. May lead to additional copies of genes.

Point mutations are single base pair alterations in DNA.

  • Missense: the original codon is replaced by a different amino acid codon
  • Nonsense: an amino acid codon is converted into a termination codon.
  • Silent: the amino acid codon is unchanged.

The Ames test was developed in the 1970s by Bruce Ames and colleagues at the University of California, Berkeley. The original screening test used bacteria to determine the mutagenesis of a given chemical compound. Versions of the test using mammalian cells and yeast have sinve been developed. 

Cancer is characterized by the malignant and abnormal growth and division of cells that can spread beyond the tissue of origin. There are over 100 different types of cancer, and they vary widely in their malignancies, causes and origin. Genes that regulate cell growth, division, differentiation, apoptosis (regulated cell death), signaling etc. are often transformed in cancer cells. When mutations target these genes or their regulatory sequences, it can lead to the production of altered proteins that can no longer function properly. The majority of cancers can therefore be traced to environmental factors promoting mutations and inherited defective genes.

This investigation uses a strain of S. cerevisiae that is deficient in a number of DNA damage repair mechanism and is sensitive to UV-induced damage. The strain was developed by Dr. John Game at the University of California at Berkeley and can be obtained through the Carolina Biological Supply Company (online at www.carolina.com). The gene URA3 encodes the enzyme ODCase (orotidine 5’-phosphate decarboxylase) that is involved in the synthesis of pyrimidine ribonucleotides. When 5-FOA (5-Fluoroorotic acid) is added to the feeding media (SD 5-FOA), ODCase will catalyze the conversion of 5-FOA to 5-fluorouracil, a toxic compound, leading to cell death. Other media used in this investigation are the complete medium called YPD (yeast extract peptone dextrose) and a minimal medium SD-ura that lacks the pyrimidine uracil necessary for growth. In this investigation, the UV sensitive strain is maintained in the SD-ura medium. This forces the yeast to both maintain the URA gene and later collect mutations in the URA gene that will allow it to survive the 5-FOA medium.

Note: 5-FOA can be difficult to obtain (reagents and plates can be bought online, but it may be simpler to contact a yeast lab). The following investigation was performed with equipment and scientists from a yeast labortory. An alternative to 5-FOA would be to use UV light (strong sunlight or a lamp) to screen for mutagens in UV-sensitive yeast, although the effect may not be as pronounced. An example of a lesson exploring UV-sensitive yeast can be found here: http://www.scienceteacherprogram.org/biology/Shah04.html

Another option is to obtain the D7 strain of S. cerevisiae (ATCC has frozen stocks available: http://www.atcc.org/Products/All/MYA-4852.aspx). D7 cells are not able to grow on synthetic media lacking tryptophan. Furthermore, when mutations are induced at the ade2 locus, the colonies change in color from white to red. This allows for visual screening and isolation.

An advanced lesson plan using D7 is described here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2104512/

Agar plate protocol: http://openwetware.org/wiki/Pour_YPD_plates

5-FOA plating protocol: http://mcb.berkeley.edu/labs/koshland/Protocols/Media/5foa.html

Culture tips here: http://homepages.gac.edu/~dahlseid/CHE360/Lab/culturetips.html

Lesson Implementation / Outline

Introduction: 

Before the start of the lesson:

- Keep the YPD plates at room temperature.
- Keep the 5FOA plates at 4°C.
- (Optional) For demonstration purposes, have normal and mutant (URA) yeast plated on 5FOA and YPD plates.
- The day before the mutagenesis labs (day 2 and day 4) start an overnight culture of the UV-sensitive yeast. Note that you’ll need about 400 ul of saturated culture per student pair. Grow the cells in SD­-Ura, so that the cells are forced to keep the URA gene to function; otherwise you can get a high background of spontaneous loss of URA without any mutagenesis.
- Bring a variety of compounds such as hair dye, tobacco, soda, milk, oil etc. Notes: 1. avoid toxic compounds! 2. Compounds that dissolve into solution are easier to handle.
- Set up microscopes.

Activity: 

Preparation

- Print out the worksheets in “cell_microscopy.docx”
- Presentation: “mutagenesis_day1.ppt”
- Take out a few plates of yeast.

Lesson 1 - (70 min class) ­- Introduction

1. Start the lesson in the room where the microscopes are (Note: this might be distracting for the students). Have worksheet #1 and #2 (found in “cell_microscopy.docx” attached below) ready to hand out. The lesson follows the presentation.

2. Brainstorming

- Ask the students what they think cancer is. Give the students 5 min to answer the question. Use a non­-biased approach to pick 3­-4 students to read their response.

3. Introduction to cancer

- Play “So true, so false” using common misconceptions or facts about cancer

- Short presentation about cancer epidemiology and causes

6. Introduction to yeast

- Yeast is present in our daily lives. Some do not cause us harm such as those found in the form of food (bread) and beverages (beer, wine, kombucha). Other types of yeast can cause diseases or cause us to get sick (Candida, yeast infections).

- Yeast is a model organism used in research. What is a model organism?

- Pass out cotton swabs and slides. Demonstrate how to place a drop on a slide, staining and coverslipping. Note: The iodine stain is used to visualize the cells more clearly.

7.  Worksheet #1 (in cell microscopy document):

Materials

- Glass microscope slides
- Glass cover slips
- Paper towels or tissue
- Iodine solution
- Plastic pipette or dropper
- Cotton swabs

Method for cheek cells

- Take a clean cotton swab and gently scrape the inside of your mouth.
- Smear the cotton swab on the centre of the microscope slide for 2 to 3 seconds.
- Add a drop of iodine solution and place a coverslip on top.
- Remove any excess solution by allowing a paper towel to touch one side of the coverslip.
- Place the slide on the microscope, with 4 x or 10 x objective in position and find a cell. Then view at higher - magnification.

- Worksheet #1 (in cell microscopy document): What do the cells look like? Observe and draw.

- Once completed, give the students a plate with yeast colonies. Have the students complete Worksheet #2 (in cell microscopy document).

8. Worksheet #2 (in cell microscopy document):

Method for yeast cells

- Take the microscope slide and put a small drop of water in the middle
- Take the yeast plate and pick a single colony by gently touching with a toothpick. Note, you might not see anything on the toothpick, but because yeast cells are so small, you probably picked more than enough!
- Smear the toothpick tip in the water droplet of the microscope slide for 2 to 3 seconds.
- Coverslip.
- Remove any excess solution by allowing a paper towel to touch one side of the coverslip.
- Place the slide on the microscope, with 4 x or 10 x objective in position and find a cell. Then view at higher magnification.

­ Ask them to make a table in their notebooks and compare the mammalian cells to the yeast cells. How are they different? Similar?

9. Introduction to mutagenesis

- Introduce the concept of mutagenesis and mutations.

­ Introduce the different types of mutations and ask the students whether they are “good”, “bad” or “neutral”.

­ What are mutagens?  Mutagens induce mutations in genetic material.

­ Make the students name mutagens ­ go around the room, so that every students gets a chance to talk. How are mutagens related to cancer? How can mutagens influence natural selection?

10. Yeast as a model system to study mutagenesis

- Explain the Ames test and introduce the investigation.

- Introduce and explain the 5-FOA plates and the yeast URA (UV-sensitive) mutant.

- Explain the goal of the lab and the expectations: yeast will be exposed to different substances and plated to see if they can grow on 5-FOA plates. A mutagenic substance would be expected to induce mutations in the yeast URA gene that will allow them to survive on the 5-FOA plate. As a control, some yeast will be left untreated.

- Ask the students to pick a mutagen they want to test (soluble, non-toxic). Discuss guidelines (no toxic compounds, ease of handling etc.) Have them bring the item next class or use a sign-up sheet to use the materials provided.

Day 2 (70 min class) - Mutagenesis Lab

Preparation

- Print out “Questionnaires.docx”
- Presentation: "day2.ppt”
- Move 5-FOA plates to room temperature.
- Eppendorf tubes filled with yeast for each student.

­

1. Review the previous lesson.

Ask the students what mutagens they have brought. Have the students pair up.

­ Offer a variety of mutagens (diet coke, lysol, salt, pepper, etc) if students want to switch or if they have forgotten to bring one. Check off mutagens brought in before use.

Give students the worksheet and Questionnaire #1 (within Questionnaire.docx). Remind the students that they will give presentations at the end of the series based on their experiments so they should take good notes and fill out the questionnaires thoroughly!

2. Student investigation

Clean hands and work area.

  • Wash your hands with soap and water.
  • Wipe your hands and your work area with alcohol and a paper towel.

- Acquire a Petri dish from the instructor and label it using a waterproof marker.

  • Write your name in small letters around the outside edge of the bottom of the dish.
  • Draw lines on the bottom of the dish to divide it into 3 parts.
  • Label one area “control”. What is a control and why do you need it? What is your control?
  • Label each area on the bottom of the dish.
  • Write in small letters around the edge.

Pick two mutagens.

  • Discuss with your partner what mutagens you want to test. You can pick from the collection provided by your instructors or come up with your own! Note: if you want to test your own mutagens, you should first talk to your instructor!
  • Label the remaining two thirds of the Petri dish with the names of the mutagens you chose.

- Acquire yeast from the instructor.

  • Swirl the container of yeast.
  • Add water (2 drops) to the negative control yeast.
  • Add your first mutagen of choice to the second tube with yeast. Note: consult your instructor about how much mutagen you should add! (Start with 2 drops of liquid or a toothpick scoopful of powder/solid)
  • Add your second mutagen of choice to the third tube with yeast. Note: consult your instructor about how much mutagen you should add!
  • Incubate the yeast for 10 min either at room temperature or at 30°C.
  • Acquire a questionnaire from your instructor and fill it in while waiting. Return your completed worksheet to your instructor when you finish!
  • After the incubation, transfer the yeast solution with a sterile pipet on the media on the corresponding third of the plate.
  • Gently spread the solution with a toothpick.
  • Let it dry for 5 min.
  • Return the plate to your instructor.
  • Clean hands and work area.

3. Finishing

- Collect the plates and place at 30°C for 3-4 days to allow the yeast to grow. Move plates to 4°C to stop growth. 

Day 3 (55 min class) – Data Analysis

Preparation

- Print out “Chi_analysis.docx” and “coin_flipping_activity.docx”
- Presentation: “day3.ppt”
- Bring enough pennies to share with the class (or other coins, such as a weighted or trick coin) 

1. Introduction

- Review the experiment and what the expected results are based on their setup. The control plate should not have any colonies; the non­mutagen plates also should not have any colonies; mutagen plates should have colonies.

- Discuss why we might see colonies on the control plate.  Some possibilities include random mutagenesis, cell stress inducing mutations, human error.

- Go over the following questions:

  • If we see 0 colonies on the control part of the plate and 20 on the experimental, do you think the substance is mutagenic?
  • What If we see 1 colony on the control part of the plate and 20 on the experimental?
  • What if we see 19 on the control plate and 20 on the experimental?
  • What if we see 15 on the control plate and 20 on the experimental?

- Introduce statistical analysis and why it is useful. Discuss how scientists use statistical tests to determine the significance of their data. Statistics tell us whether a difference is real or occurred by random chance (ie: Chi Square test). 

- Introduce the Chi Square test

- Define: a null hypothesis, significance.

2. Coin Flipping activity

 - Coin Flipping activity as an analogy: Go through the first section of the worksheet “Worksheet 1 Coin Flipping Activity” as a group. Pass out coins.

- Take a quarter and flip it 20 times. Record the number of heads and tails.
- What is the expected number of heads and tails, assuming that it is equally likely to get heads and tails? 10 / 10
- Calculate the difference between observed (first table) and expected (second table) number of heads and tails.
- Square the obtained numbers.
- Divide the number by the expected number of heads or tails.
- Now add the two numbers together; this is chi square!
 - Is the obtained number greater or less than 3.84?

**3.84 is a cutoff that tells you whether the difference you observe is significant or if it happens by chance. Chi squares greater than 3.84 are significant. Chi square values of less than 3.84 are not significant (the difference happens randomly/by chance).

- Is your coin fair or biased? (Bringing in a weighted coin and doing the exercise is also an option)

3. Data analysis

- Pass out the plates, and ask students to count their colonies. Record results on  “Worksheet 1 Colony counting” (found in Chi anlysis document). The worksheet can be completed as homework instead depending on time available. Determine if the results obtained were "meaningful."

- If you tested two mutagens (coffee and splenda) and their combination (coffee + splenda), do the worksheet twice for each mutagen (coffee versus control and then splenda versus control).

- What is the null hypothesis?

- Count the number of colonies on your control part of the plate and the part with the substance tested (coffee, mascara, shampoo, etc.).

- What is the expected number of colonies in the control and the test, assuming that that the yeast is equally likely to grow on either side? Note that this number will be the same for the test and the control and will be ½ of all the colonies that you’ve counted (for example, if you count 40 total colonies on your test and your control sections, the number will be ½ of 40 = 20 colonies).

- Calculate the difference between observed (first table) and expected (second table) number of colonies.
- Square the obtained numbers.
- Divide the number by the expected number of colonies.
- Now add the two numbers together; this is chi square!

- Is the obtained number greater or less than 3.84?

- Can your substance cause mutations in yeast?

Day 4 (70 min class) - Protocol modification

Preparation

- Print out “Chi_analysis”, “Modify_Protocol_Procedure.doc”
- Presentation: “sep_modify_protocol_day4.ppt”
- Have beakers, pipets, plates, tubes of yeast and mutagens available. 

1. Review

- Review chi square analysis. Address students’ confusion about null hypothesis and what the chi square value means with respect to the substance being a mutagen or not. Go through a couple of examples on the board.

2. Introduce the concept of modifications to protocol to test new questions.

- Ask the class to come up with some ways they could make a substance more or less mutagenic. Examples include: increasing or decreasing the amount of the substance they add to the yeast, changing the incubation time, mixing two substances together, etc.

- Pass out materials: plates, tubes with yeast, substances. Make sure to have a couple beakers with water and transfer pipets that can be brought around to the tables.

- Ask students to perform the experiment following the instructions on the worksheet (Modify_Protocol_Procedure). Have them fill out “Questionnaire #2” (found in questionnaire.docx).

3. Modified mutagenesis lab 

- Clean your hands and work area. Clean your hands with hand sanitizer
- Acquire a Petri dish from your instructor and label it using a waterproof marker.
- Write your name in small letters around the outside edge of the bottom of the dish.
- Draw lines on the bottom of the dish to divide it into 3 parts.
- Label one area “control”.  What is a control and why do you need it? What is your control?
- Label each area on the bottom of the dish. Write in small letters around the edge.
- Discuss with your partner what mutagen you want to test. You can test the same mutagen as last time or pick a different one from the collection provided by your instructor. Note that if you want to test your own mutagen, you should first talk to your instructor!
- Label one of the remaining two thirds of the Petri dish with the name of the mutagen you chose.
- Modify the protocol: for the last third of the Petri dish you will modify the protocol in a way that will make your mutagen stronger or weaker!

Option A: Yeast Protection

- Discuss with your partner how you can modify the described about mutagenesis with your substance of choice in order to make it less mutagenic. You can:

  • add less mutagen (less than 2 drops of liquid or a scoop of powder/solid)
  • incubate for less than ten minutes
  • add another substance in addition to the first one
  • protect from sunlight (with foil, sunscreen etc.)
  • use your imagination!

- Write down the modifications you’ll make to the protocol. 

Option B: Stronger mutagenesis 

- Discuss with your partner how you can modify the described about mutagenesis with your substance of choice in order to make it more mutagenic. You can:

  • add more mutagen (more than 2 drops of liquid or a scoop of powder/solid)
  • incubate longer than ten minutes
  • add another mutagen in addition to the first one
  • expose to sunlight
  • use your imagination!

- Write down the modifications you’ll make to the protocol. Acquire yeast tubes from your instructor.
- Acquire yeast tubes from your instructor.
- Label the tubes and the pipettes -> A: control, B: your mutagen, and C: modified mutagen.
- Swirl the container of yeast.
- Add water (2 drops) to the control yeast (tube A).
- Add your mutagen of choice to the second tube with yeast (tube B). If your mutagen is liquid (shampoo, hot sauce, etc.), add 2 drops. If your mutagen is very viscous, powder or solid (toothpaste, splenda, etc.), pick a small amount with a toothpick and add to the yeast container.
- If you are modifying the amount of mutagen added to the third container (tube C), add the altered amount (more, less, in combination with another mutagen, etc.). Otherwise, add the same amount of mutagen as in tube B.
- Incubate the yeast for 10 min.
Note: if you are altering the incubation time for tube C (incubate for more or less than 10 min), incubate that tube accordingly!  

- Complete Questionnaire #2 (found in questionnaire.docx) while waiting. Return your completed worksheet to your instructor.
- After the incubation, transfer the yeast solution with a sterile pipet on the media on the corresponding thirds of the plate.
- Gently spread the liquid with a toothpick.
- Let it dry for 5 min.
- Return the plate to your instructor

Questionnaire #2 (found in questionaire.docx)

- Did you choose to protect the yeast or mutagenize them more?
- What modification are you going to make?
- Why?
- How is this modification going to affect your results? Do you expect more or less colonies? Why?

4. Finishing up

- Collect the returned plates and incubate them at 30°C for the next 3-4 days to allow the yeast to grow. Store at 4°C beyond that.

Day 5 (40 min class) – Data analysis 2

Preparation

- Print out “Chi_analysis.docx”, "Worksheet 2 Colony Counting (pg 2 in "coin flipping activity" document) and “guidelines_for_presentations.docx”
- Take plates out to room temperature. 

1. Review the experiment and what the expected results are based on the setup.

- The control plate should not have any colonies; non­mutagens also no colonies; mutagens should have colonies on the plate; modified mutagens may have more or less colonies depending on modification made.

2. Data analysis.

- Pass out the plates, and ask students to count their colonies. Record results on  “Worksheet 2 Colony counting” (pg 2 in "coin flipping activity" document). Determine if the results obtained were "meaningful."

- For this part of the worksheet, consider only the modification (soy sauce + toothpaste, more shampoo, etc.) and the part on the plate without the modification (only toothpaste, one scoop of shampoo, etc).

- What is the null hypothesis?  
- Count the number of colonies on your modified part of the plate and the part without the modification.
- What is the expected number of colonies in the control and the test, assuming that that the yeast is equally likely to grow on either side? Note that this number will be the same for the test and the control and will be ½ of all the colonies that you’ve counted (for example, if you count 40 total colonies on your test and your control sections, the number will be ½ of 40 = 20 colonies).
- Calculate the difference between observed (first table) and expected (second table) number of colonies.
- Square the obtained numbers.
- Divide the number by the expected number of colonies.
- Now add the two numbers together; this is chi square!
- Is the obtained number greater or less than 3.84?
- Did your modification make your substance more or less mutagenic?

3. Discuss as a class the format and content of their presentations. This could be a Powerpoint presentation, a short talk or a poster.

- Hand out the guidelines (under guidelines_for_presentations.docx). Have students group up.
- Students will be expected to answer the following questions in their presentations:

Introduction
- What are mutations and why are they interesting? Why do you want to test if a substance is mutagenic?
Hypothesis
- What substance did you choose and did you expect your substance to make mutations? Did you expect to see colonies on the plate?
Procedure
- How did you do your test?
Results
-  How many colonies did you count on the control part and on the test substance part of the plate? Was your substance a mutagen according to your statistical analysis (chi square)?
Modifications
-  What did you change and why? What were the results?      
Conclusions
- Were the substances you tested mutagenic? If you could do another experiment, what would it be and why? 

- Check for student understanding: do all mutations lead to cancer? Why or why not?
- How could you use the Ames test in your community? (pollution tests: local water quality for example)

Lesson 6 (70 min class) - Student Presentations!

Format: 5 minute presentations + 1­2 min questions from the class.

Wrap-up / Closure: 

Questions to discuss:

- What was your final result?
- What would you do next?
- What would you change if you did this again?
- What questions do you now have? 

Extensions and Reflections

Extensions and connections: 

Links of interest:

The following could be a good follow up to this lab:
http://teach.genetics.utah.edu/content/begin/dna/sunscreen%20teacher.pdf

L.H. Hartwell’s Yeast: A Model Organism for Studying Somatic Mutations and Cancer
http://www.nature.com/scitable/topicpage/l-h-hartwell-s-yeast-a-model-808

More information on the Chi square test:
http://www.radford.edu/rsheehy/Gen_flash/Tutorials/Chi-Square_tutorial/x2-tut.htm

AttachmentSize
mutagenesis_day_1.ppt3.19 MB
day_2.ppt4.16 MB
day_3.ppt722 KB
sep_modify_protocol_day4.ppt280.5 KB
cell_microscopy.docx52.27 KB
questionnaires.docx107.46 KB
chi_analysis.docx90.25 KB
coin_flipping_activity.docx68.19 KB
modify_protocol_procedure.docx143.47 KB
guidelines_for_presentations.docx69.12 KB
NGSS Topics
NGSS Disciplinary Core Ideas
NGSS Performance Expectations
NGSS Performance Expectations: 
HS-LS1-1
HS-LS3-2
HS-LS4-2
NGSS Science and Engineering Practices
NGSS Crosscutting Concepts
NGSS Crosscutting Concepts: 

Standards - Grades 9-12 Biology

Genetics: 
c. Students know how mutations in the DNA sequence of a gene may or may not affect the expression of the gene or the sequence of amino acids in an encoded protein.

Standards - Grades 9-12 Investigation and Experimentation

Investigation and Experimentation: 
a. Select and use appropriate tools and technology (such as computer-linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data.
b. Identify and communicate sources of unavoidable experimental error.
c. Identify possible reasons for inconsistent results, such as sources of error or uncontrolled conditions.
j. Recognize the issues of statistical variability and the need for controlled tests.