Transcript Lab 13 Substitute * Yeast Catalase Activity

Lab 7 – Heredity
Is there a fly in here?
Note: This lab is another “old lab” from the previous
manual, however you’ll need to write it into your lab
notebooks as an informal report (not a packet).
The Background
• Drosophila melanogaster, also known as a fruit fly (or
gnat), is a very common model for genetic studies.
• Its entire genome has been sequenced and thus we
can create entire lineages of Drosophila by
manipulating their genes.
• We’re going to explore this concept further by having
you cross fruit flies with particular traits.
• Here are some common ones that have been made
over the years through genetic modification.
Phenotypes
Vestigial Winged
Wild Type
Curly Winged
White Eyed
Ebony
Yellow
Antennapedia
(head-legs)
Eyeless
Orange Eyed
http://www.unc.edu/depts/our/hhmi/hhmi-ft_learning_modules/fruitflymodule/phenotypes.html
The Variants
Wild Type
• Normal Wings vs. Vestigial Wings
https://tbsgeneticsa.wikispaces.com/file/view/wild_vg.jpg/188678709/wild_vg.jpg
http://www.schooltr.com/Teacher_Resources/Lesson_Plans/Lesson_Plan_images/Drowsy_Drosophila15.jpg
Vestigial Winged
The Variants
Wild Type
• Normal Eyes vs. White Eyed
http://images.fineartamerica.com/images-medium-large/wild-and-white-eyed-fruit-flies-photo-researchers.jpg
http://www.brain.riken.jp/bsi-news/bsinews30/files/network0101-big.jpg
White Eyed
Lab Setup
• For today – we’ll call this Day 0 – you need to do this:
– Decide on which cross you want to do and let me know when
I come to your table.
– Set up your fruit fly tubes.
• Get a plastic tube and a foam stopper.
• Use a felt-tip pen to write your group’s names on the tube (masking
tape available) as well as the genetic variant (wild type and vestigial
wings/white eyes).
– Don’t forget to decide which gender will be which.
• I will add a small layer of food (called white media) – you add a
roughly same-sized layer of water on top until it dampens but isn’t
runny.
• Include, if possible, a piece of plastic mesh.
– Plug the tube and place it by the windows.
Background Drosophila Information
• Flies have been anesthetized by being refrigerated
overnight, as have been the petri dishes in which
they will be observed.
– Being ectothermic, fruit flies’ metabolisms slow down to
the point that they cannot move.
• Your goal in these initial observations is to get good
at sexing males and females.
– Use the dissecting scope (stereoscope) – zoom is above
the stage.
– Make sure everyone in your group gets good at this.
How to Sex a Fruit Fly
• Differing Characteristics:
• ♂
– Dark, rounded abdomens
• ♀
– Striped, pointed abdomens
• The “point” is an ovipositer –
the egg-laying organ.
• Males also have a “sex
comb” on their front legs,
but it’s hard to spot.
http://www.stonybrook.edu/commcms/biology/images/Drosophila%20Male%20Female.jpg
http://ls.tcu.edu.tw/mdlin/Userdata/Image/male%20and%20female.jpg
Background Drosophila Information
• The complete Drosophila life
cycle takes 10-12 days on
average.
– See lab sheet for more
information.
– Disclaimer: I will refer to
adults as “hatching,” but in
reality they metamorphose
from pupae.
• Mating, as you might
imagine, is hard to control.
• Furthermore, females store
sperm and only release it
when they lay eggs.
Drosophila Life Cycle
http://www.flinnsci.com/store/catalogPhotos/FB0361cat.jpg
Background Drosophila Information
• Since females can store sperm, we can’t control
crosses if they’ve already had a chance to mate.
– We need to ensure females have had no such opportunity
and that they can only mate with males we’re crossing with
them.
• In order to control the experiment, then, we need to
use virgins.
– The adults we will be observing today as practice will be
killed afterward.
• Or saved. We’ll see. But they always need to be removed before…
– …next class, when we’ll separate the newly-hatched flies
before they reach maturity and mate with one another.
Lab Sequence
• Day 0:
– We did this already – it was our initial set up of food
containers.
• Day 1:
– That’s today. We’re practicing sexing flies and identifying
phenotypes.
• Work quickly while observing flies. You may keep the lid on your
petri dish if it makes you feel more comfortable.
– Adults removed, leaving their eggs to hatch and form our P
generation.
• Seems like a good time to record this stuff in your
notebooks.
Lab Sequence
• Day 2:
– Set up our crosses by introducing the P generation parents to
one another.
– You’ll need about 3-4 males and 3-4 females from the newlyhatched individuals.
• Make sure you keep it consistent with gender – only one gender for
each trait.
– As in, “wild type females with white-eyed males,” for example.
• These pairs will [hopefully] breed and give rise to the F1 generation.
– Of the ones I tap onto your petri dish, separate the
individuals you need using a Q-tip.
• I need the rest back ASAP. Get the lid on the petri dish in case they
wake up.
Lab Sequence
• Day 3:
• Change the “3” according to whichever day it is.
– Kill the P generation adults. They’ve laid the F1 eggs.
• Day 4:
– Observe the F1 generation and record your data.
• How many individuals, how many male/female, how many
male variant, how many female variant, et cetera.
– Select 3-4 pairs of males and females like last time.
• You pick the traits to go with the sex.
• It can be different from last time (if you want).
– They’ll give rise, ultimately, to the F2 generation.
Lab Sequence
• Day 5:
• Change the “5” according to whichever day it is.
– Kill the F1 generation adults. They’ve laid the F2 eggs.
• Day 6:
– Observe the F2 generation and record your data.
• How many individuals, how many male/female, how many
male variant, how many female variant, et cetera.
• Day 7+:
– Continue observing the F2 generation and increasing
the sample size.
Lab Sequence [General]
• Day 0: Set up tube.
• Day 1: Practice sexing flies, kill the stock adults.
• Day 2: Add new immature stock adults to your own
tubes – they’re the P generation.
1 Week Intermission
• Day 3: By now, P adults have laid eggs.
– Kill P adults, wait for hatching. Set up new tube.
• Day 4: Analyze the resulting (F1) phenotypes. Cross.
1 Week Intermission
• Day 5: By now, F1 adults have laid eggs.
– Kill F1 adults, wait for hatching.
• Day 6: Analyze the resulting (F2) phenotypes.
• Day 7+: Continue observing/collecting F2 flies.
From F1 to F2
• On our first day of the transition between
generations, we’re going to count all individuals
from F1 – the ones currently in the tubes.
– And kill them.
– You’ll anesthetize them and then dump them into a
petri dish with a thin layer of ethanol.
• Next class, we’ll select some newly-hatched
individuals and make them the F2 generation,
placing them into fresh tubes with new food.
• For this first round of F1 data, record sex and
phenotype of all individuals.
From F1 to F2
• When we move to the F2 generation, select a
“handful” of flies and record sex/phenotype
information.
• If you have the ability and choice, try to make
males one phenotype and females another.
– Either way, be sure to record this info.
Analysis and Conclusion Details
• Believe it or not, there’s room for a statistical test.
• Following the analysis of the F1 generation,
develop a hypothesis for how the trait you’ve
selected is inherited.
– Is it autosomal dominant, autosomal recessive, sexlinked (dominant or recessive)?
• For each mating generation (P and F1), perform a
Punnett square and calculate the probabilities of
each phenotype found in the offspring.
– This is your null hypothesis.
Analysis and Conclusion Details
• So you’ll have expected amounts of offspring
phenotypes from the Punnett Squares, but then you’ll
also have actual recorded numbers of offspring
phenotypes that formed.
– What test does it sound like you’re going to use?
– A chi-squared test! Yay you.
• Given the total actual amount of offspring that formed,
compare observed (in the vial) phenotypes to expected
(from the Punnett square) phenotypes.
– Degrees of freedom? That depends.
– Determine degrees of freedom from your Punnett square
results.
• Remember, it’s possible outcomes minus one.
The Role of the Null Hypothesis
• Just for the record, in this case, you “want” to
accept your null hypothesis.
• Not having a difference from your Punnett
square means your hypothesis of inheritance
mode is correct.
– So accepting the null hypothesis, in this case,
means you also accept your own hypothesis, and
vice versa.
Analysis and Conclusion Details
• For your lab notebook, put the chi-squared test
(and the work) in the Analysis section.
• Put the discussion of the meaning of your chisquared results in the conclusion.
– Can you accept your null hypothesis?
– Do the data not really line up with any conceivable
pattern of inheritance?
– Could there actually have been a different kind of
inheritance at work?
– Did any parent fruit flies accidentally stick around?
– Could someone have mixed up male/female?
The Lab Report
• Put the whole thing together according to the
informal lab report rubric.
• The report will be due one week following the
conclusion of the experiment.