Canavanine Lab
Summary |
A laboratory screen is often used to enhance experimental results that are not easily
identified in the field. In this experiment, we use a canavanine screen to observe and
identify UV-induced mutations. Students will conduct a series of dilutions of wild-type
strains ofSaccharomyces cerevisiae and plate them on YPD agarose plates containing
canavanine and a control plate with no canavanine. Students will be able to explore the
concepts of mutations and their detrimental effects from environmental
factors. Students will compare the results for both groups and estimate the rate of
mutation rates between canavanine and UV irradiation. This lab is designed to be
inquiry-based, where students have the flexibility to explore the light intensity and
time. Either a UV lamp or the natural sunlight can be used to irradiate.
Please provide a brief summary of your lesson, including its broader context within the curriculum.
What will students be doing? What questions or subjects will the be exploring? What datasets (if
applicable) will they be using? What skills will they be developing?
Background Information
Canavanine is found naturally in plant seeds such as alfalfa and functions as an aminio
acid analog that can take the place of arginine in proteins. Canavanine enters the cell
via an arginine permease controlled by the CAN1 gene. Once in the cell, arginyl-tRNA
synthetase binds canavanine to tRNA in the place of arginine. Canavanine is then
incorporated instead of arginine into a protein resulting in a faulty protein.
A canavanine screen is commonly used in yeast studies to measure and quantify
mutation rates. UV light causes mutations in the DNA of yeast. When wild type yeast
cells are plated on a plate with canavanine, the cells will die from protein translation
errors caused by canavanine taking the place of arginine. When yeast cells are exposed
to UV light, their DNA will be randomly mutated and the rate of these mutations can be
quantified by observing how many yeast colonies can form on a canavanine plate. If the
CAN1 gene is mutated during UV exposure, the yeast cells will no longer take-up
canavanine thereby protecting the cell from canavanine induced protein translation
errors.
Key Concepts
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mutations
haploid vs. diploid cells
wild-type vs. mutant
protein synthesis
o amino acids
o translation
o tRNA synthetase
• genetic screens
Objectives
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SWBAT quantify mutation rates.
SWBAT record yeast colony counts.
SWBAT produce serial dilutions.
Observe and identify
o Mutations produced in a canavanine screen
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Record
o Create charts and graphs to collect data
Demonstrate
o create charts and graphs to collect data
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laboratory skills i.e., growing and counting yeast colonies, pipetting, serial dilutions
measure volumes in
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• Communicate results
o write a formal laboratory report
o analyze data
o present findings to the class
o explain in writing how canavanine will affect the translation process
o explain how environmental factors can cause mutations
Materials
• UV light source (sun, UV-C lamp)
• Canavanine prepped plates
• YPD agarose gel
• Include any additional worksheets or resources
Procedure
1.
Include the step-by-step procedure for completing the lesson
To determine if UV irradiation induces mutations we will use a screen to detect mutants
that are resistant to canavanine.
Canavanine is a natural compound found in leguminous plants. It is an analogue of the
amino acid Arginine. Canavanine is toxic to organisms because it is mistakenly
incorporated into proteins instead of arginine. One way to become resistant to
Canavanine is to block its uptake in the cell. If the transporter for arginine is mutated in
yeast, arginine (and canavanine) from the outside can no longer be transported inside.
Therefore canavanine is no longer toxic but the cells must be able to synthesize arginine
on their own in order to survive.
Therfore selecting for canavanine resistance is an easy way to assess mutagenesis.
You will plate a lawn of the wild type and rev mutant strains onto minimal plates that
contain canavanine and no arginine. You will irradiate the plates at a UV dose that kills
about 90% and about 99% of the cells (10% and 1% survival).
You will then determine the number of colonies that can grow on canavanine with no
irradiation (spontaneous mutations) and compare it to the numbers of mutants that appear
at the low and high dose of UV.
Plate 100µl and 500µl of undiluted cells onto the Minimal agar pates with Canavanine to
create a lawn.
You will test three UV doses (50% survival, 10% survival. 1% survival)
No Canavanine-
+ Canavanine
Survival experiment
Mutagenesis experiment
For WT strain
For WT strain
100 µl of 10-5 dilution
100 µl of undiluted culture
100µl of 10-6 dilution
100 µl of undiluted culture
For WT strain
For WT strain
100 µl of 10-5 dilution
100 µl of undiluted culture
100µl of 10-4 dilution
100 µl of 10-1 dilution
No UV
UV
Canavanine
Time
UV Irradiation
Wild-Type
Number of Colonies
Assessment
• Performance—what will students do during the lesson to demonstrate understanding?
• Product—what will students produce to demonstrate understanding?
• Assessment should be directly related to the lesson objectives
• Assessment rubrics that you would use in the classroom are also helpful
Additional Resources
How to make a canavanine plate
Got time?
If you have time before your presentation, it would be helpful to provide
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Relevant content standards—National Science Education
Standards: http://www.nap.edu/readingroom/books/nses/html/6a.html
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Key concepts, according to the AAAS benchmarks, which provide a framework for
K-12 expectations:http://www.project2061.org/publications/bsl/online/bolintro.htm
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Gene mutation in a cell can result in uncontrolled division called cancer.
Exposure of cells to certain chemicals and radiation increases mutations and
thus the chance of cancer. 5C/H6