Yeast gene expression lab using
b-galactosidase vectors that can be completed in
one 2 hour laboratory session.
Stephanie C Schroeder, PhD
Assistant Professor, Dept. of Biological Sciences
Webster University, St. Louis, MO
Background
BIOL 3050/3051 is a 16-week sophomore level Genetics
Lecture and Laboratory course.
Both lecture and lab are taught by same instructor (me)
The lab meets 3 hours, once per week.
The lab classroom is shared between other classes, so is not
accessible to students outside of the lab period.
Lab Schedule
Title/Topic
Meiotic Recombination
Monohybrid/Dihybrid
Sex Linked Traits
Genetic Linkage
Transformation
(importance of CEN/ARS)
Population Genetics
DNA repair (UV mutagenesis)
Mitochondrial DNA
Gene expression
Model Organism
Sordaria
Corn/Drosophila/Yeast
Drosophila
Drosophila
Yeast
Drosophila
Yeast
Human
Bacteria and Yeast
Background
This lab was designed to demonstrate two
principles of gene expression in eukaryotes
1. Differences between positive and negative
acting elements
2. Synergy with multiple promoter elements.
• Title: Eukaryotic Gene Expression
• Purpose: To perform b Galactosidase assays
on yeast strains containing plasmids with
different promoters to determine the
activity of these promoter fragments.
Strains were generated in lab of Tony Weil, at Vanderbilt University.
Constructs for Part One
Wild Type
T
A
x
A
A
B
lacZ
C
Deletion ElementA
T
B
x
B
lacZ
C
Deletion Element B
T
C
lacZ
Results from Original Paper Identifying
Three Cis-Acting Elements
A
B
C
Part Two: Synergy
Element C was subcloned into the XhoI site at -178
1 copy
2 copies
3 copies
4 copies
Synergy
Pre Lab Prep
Week before : (I have students make the Z buffer and ONPG)
1. Streak out strains from glycerol stock onto Sc-ura plates
2. Make Z buffer, ONPG (wrap in foil), Na2CO3
Day before lab: (I do)
Inoculate 5 ml cultures of Sc-ura with strains
Morning of lab:
1. Read Absorbances at 600 nm for overnight cultures
2. Aliquot yeast into 1.5 ml tubes for students
3. Aliquot SDS, Chloroform, Na2CO3
Protocol For Students (They work in pairs)
You have 8 tubes (labeled 1-8) which contain 0.2 mL each of the yeast strains listed in
Table I.
At the beginning of your lab session, I will tell you the A600 for each strain, to enter into
your Table I.
1. Vortex the cultures to mix the cells..
2. Add 0.8 ml Z buffer to each tube. (P-1000 set to 080)
3. Add 50 ul chloroform to each tube (P-200 set to 050)
4. Add 20 ul 0.1% SDS to each tube (P-20 set to 200)
5. Vortex each tube for 10"
6. Add 200 ul ONPG (1mg/ml in Z buffer +BME). Vortex the tubes to mix.
7. Time reaction at Room temperature (30 minutes from time added to tube 1)
8. Stop with 0.5ml 1M Na2CO3.
9. Pellet 5 minutes in eppendorf centrifuge
10. Carefully transfer upper layer to a cuvette (chloroform will “etch” or dissolve the
cuvettes)
11. Read the Absorbance at 420 nm (blank with tube that says Blank))
Taken from Cold Spring Harbor Yeast Genetics Lab Handbook (1989)
Cell culture
Table I:
Absorbance
at 600
Volume (mls)
Time
1. No Promoter
0.2
30
2. Wild Type
Promoter
0.2
30
3. Deletion
DNA Element A
0.2
30
4. Deletion
DNA Element B
0.2
30
5. CYC1 vector alone
0.2
30
6. CYC1 +
2 copies element C
0.2
30
7. CYC1 +
3 copies element C
0.2
30
8. CYC1 +
4 copies element C
0.2
30
Absorbance at 420
Activity Units
Calculate activity using the following formula:
Activity Units= Absorbance at 420 nm x 1000
= Velocity/mL
A600 x 30 minutes x 0.2 mL (200uL)
Write-up/ Conclusions
Construct ONE graph (bar graph) with the Activity Units for each construct on the Y
axis and the names of the promoter constructs on the X axis. Use this graph to answer
the following questions.
Typical results from student
experiments
300
Activity
250
200
150
S
100
50
0
No
vector
Full
Deletion Deletion
Length
A
B
CYC1
CYC1 + CYC1 + CYC1 +
2C
3C
4C
Questions:
1.In the equation to determine the b-galactosidase activity for our samples, why do we
divide by the Absorbance at 600 nm?
2.What fold difference is there in b-galactosidase activity between sample 1 (empty vector)
and sample 2 (full length promoter)?
3.Do you think the DNA sequence Element A is a positive acting element or a negative
acting element based on the difference between sample 2 (full length) and sample 3 (deletion
of Element A)? Explain your rationale.
4.Do you think the DNA sequence Element B is a positive acting element or a negative
acting element based on the difference between sample 2 (full length) and sample 4 (deletion
of Element B)? Explain your rationale.
5.Samples 5-8 are constructs in which I inserted sequences from the TBP promoter into a
DIFFERENT promoter (CYC1) to see their effect in a heterologous system. Based on the
differences between samples 6,7, and 8 (2, 3 and 4 copies of Element C) compared to
sample 5 (CYC1 alone), is the sequence Element C a positive or negative acting element?
Explain your rationale
6.Based on your answers above, what do you think would happen if we put the sequences
Element A from the TBP promoter into the CYC1 promoter? Based on the activity of the
CYC1 promoter alone, do you think we could really measure a difference?
Assessment
• Classroom discussion (lecture and lab)
• Students answers to questions
• Ability to apply to lecture exam/cumulative
final