Welcome!
Teaching Evolution through Inquiry
with
Bio-Rad & Vernier
Workshop
Timeline
• Introduction
• Comparative Proteomics I : Protein Profiler
– Introduction to Kit
– Load and electrophorese protein samples
– Compare protein profiles
– Stain polyacrylamide gels
– Construct cladograms
– BLAST to construct cladograms
• Biofuel Enzyme Kit: Activity 6
– Introduction to Kit
– Extract Cellobiase from Mushrooms
– Run enzyme reaction
– Obtain quantitative data with spectrophotometry
– Compare Mushroom activities
• Look at Protein Profiler gels
• Other evolution labs brief – PV92
How does
this fit with
AP Biology?
Big Idea 1
Protein Profiler
Biofuel Enzyme
PV92 PCR
pGLO Transformation
DNA Fingerprinting
DNA
Protein
Trait
Ways to analyze
evolutionary
relatedness
Comparative Proteomics Kit
I: Protein Profiler Module
Technology
Engineering
Math
Science
Inquiry
Comparative
Proteomics I:
Protein Profiler
Kit Advantages
• Analyze protein profiles from a variety of
fish
• Study protein structure/function
• Use polyacrylamide electrophoresis to
separate proteins by size
• Construct cladograms using data from
students’ gel analysis
• Compare biochemical and phylogenetic
relationships. Hands-on evolution wet lab
• Sufficient materials for 8 student
workstations
• Can be completed in three 45 minute lab
sessions
Workflow
Set Up Protein
Electrophoresis
units
Mini-Protean
TGX Gels
Using a
Micropipette
Plunger
Two stops
1st – defines volume
2nd – ejects volume
Tip Ejector
Run Protein
Electrophoresis
Lane 1-2
Lane 3
Lane 4-8
Lane 9
Empty
5ul Protein ladder
5ul Fish samples
5ul Actin & Myosin
Standards
300V
18 minutes
Samples
Actin
• 5% of total protein
• 20% of vertebrate
muscle mass
• 375 amino acids
= 42 kD
Myosin
•Tetramer
•two heavy subunits (220 kD)
•two light subunits (15-25 kD)
•Uses ATP for muscle contraction
Muscle
Contains
Proteins of
Many Sizes
Protein
kD
Function
Titin
3000
Center myosin in sarcomere
Dystrophin
400
Anchoring to plasma membrane
Filamin
270
Cross-link filaments
210
Slide filaments
Myosin
heavy chain
Spectrin
265
Attach filaments to plasma
membrane
Nebulin
107
Regulate actin assembly
-actinin
100
Bundle filaments
Gelosin
90
Fragment filaments
Fimbrin
68
Bundle filaments
Actin
42
Form filaments
Tropomysin
35
Strengthen filaments
Myosin
light chain
15-25
Slide filaments
30, 19, 17
Mediate contraction
Troponin (T.I.C.)
Thymosin
5
Sequester actin monomers
Levels of
Protein
Organization
1o
2o
3o
4o
What’s in the
Sample
Buffer? &
Why?
• Tris buffer to provide appropriate pH
• Glycerol to make samples sink into wells
• Bromophenol Blue dye to visualize samples
• SDS (sodium dodecyl sulfate) detergent to
dissolve proteins and give them a negative
charge
Sample Prep
heat
Protein Gel
(polyacrylamide)
Separates Proteins
based ONLY on
their size
TGS Buffer
•
Tris-HCL
•
Glycine (shielding
for stacking)
•
SDS
Why Use
Polyacrylamide
Gels to
Separate
Proteins?
• Smaller pore size than agarose
– Polyacrylamide gel 20-200nm pores
– 3% agarose 40-80 nm pores
– 1% agarose 200-1200 nm pores
• Proteins are much smaller than DNA
– Average amino acid = 110 daltons
– Average nucleotide pair = 649 daltons
– 1 kilobase of DNA = 650 kD
– 1 kilobase of DNA encodes 333 amino acids =
36 kD
Can Proteins be Separated on Agarose Gels?
250
150
100
75
50
37
250
Myosin Heavy
Chain
Actin
Tropomyosin
25
Myosin Heavy
Chain
150
100
75
50
Actin
Tropomyosin
37
20
Myosin Light
Chains
15
25
Myosin Light
Chains
20
10
Polyacrylamide
3% Agarose
Retrieve Gels
Cut off wells and
foot of gel
Gel Staining
Water – 5 minutes
Stain – 1 hour
Gel Imaging
GelDoc EZ
White Digital
Bioimaging
system
Polyacrylamide
Gel Analysis
250
150
100
75
50
37
Myosin Heavy
Chain (210 kD)
Actin (42 kD)
Tropomyosin
(35 kD)
25
20
Myosin Light
Chain 1 (21 kD)
Myosin Light
Chain 2 (19 kD)
Myosin Light
Chain 3 (16 kD)
15
10
Using Protein Bands between
30 and 10 kb for analysis
Molecular
Mass
Estimation
50
45
40
37 (12 mm)
25 (17 mm)
20 (22 mm)
15 (27.5 mm)
10 (36 mm)
S iz e (k D )
35
30
25
20
15
10
5
0
0
10
20
Distance migrated (mm)
30
40
Molecular
Mass
Analysis With
Semi-log
Graph Paper
Size (kD)
100
10
0
10
20
30
Distance migrated (mm)
40
Each Fish
Has a
Distinct Set
of Proteins
Shark
Salmon
Trout
Catfish
Sturgeon
Total #
proteins
8
10
13
10
12
Distance
proteins
migrated
(mm)
25, 26.5,
29, 36,
36.5, 39,
44, 52
26, 27.5,
29, 32,
34.5,
36.5,
37.5,
40.5, 42,
45
26, 27.5,
29, 29.5,
32, 34.5,
36.5,
37.5,
40.5, 42,
45, 46.5,
51.5
26, 27.5,
29, 32,
36.5, 38,
38.5, 41,
46, 47.5
26, 27.5,
30, 30.5,
33, 35.5,
37, 39,
39.5, 42,
44, 47
26
31.5
26.5
31.0
27.5
30.0
28.5
29.1
29
28.6
30
27.6
30.5
27.1
32
25.6
33
24.7
34.5
23.2
35.5
22.2
36
21.7
X
36.5
21.2
X
37
20.7
37.5
20.2
38
19.7
X
38.5
19.3
X
Sturgeon
X
Catfish
Shark
32.5
Trout
Size
(kD)
25
Salmon
Distance
(mm)
Some of
Those
Proteins
Are Shared
Between
Fish
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Character
Matrix Is
Generated
and
Cladogram
Constructed
Shark
Salmon
Trout
Catfish
Sturgeon
Shark
8
2
2
2
2
Salmon
2
10
10
5
3
Trout
2
10
13
5
4
Catfish
2
5
5
10
2
Sturgeon
2
3
4
2
12
Shark
Sturgeon
Catfish Trout Salmon
Phylogenetic
Tree
Evolutionary tree showing the relationships of eukaryotes.
(Figure adapted from the tree of life web page from
the University of Arizona (www.tolweb.org).)
Pairs of Fish
May Have
More in
Common
Than to the
Others
Shark
Salmon
Trout
Catfish
Sturgeon
Carp
Shark
8
2
2
2
2
2
Salmon
2
10
10
5
3
5
Trout
2
10
13
5
4
5
Catfish
2
5
5
10
2
8
Sturge
on
2
3
4
2
12
2
Carp
2
5
5
8
2
11
Shark
Sturgeon
Catfish Carp Trout Salmon
BLAST
Searches
with Protein
Profiler
(Appendix B)
Uses Myosin Heavy Chain from Rainbow trout
•Pre-run blast searches in manual
•Protocol to run your own searches
Chart Differences:
0
8
8
9
9
7
11
11
13
11
Clustal-W
Analysis
(Sequence)
Biofuel Enzyme Kit
Activity 6 – a functional proteomics approach to
evolution in fungi
Engineering
Technology
Math
Science
Inquiry
Biofuel
Enzyme Kit
Advantages
• Aligns with AP Biology Learning Objectives LO
1.14, LO 1.17, LO 1.18, LO 1.19, LO 2.1, LO 2.2, LO
2.3, LO 2.4, LO 2.10, LO 4.14, LO 4.15, LO 4.18)
• Can be run qualitatively or quantitatively
• Construct and use a standard curve (mathematics
and technology)
• Determine the effects on the reaction rate by
changing:
– pH
– temperature
– enzyme/substrate concentration
• Mushroom extract activity for student run inquiry
• Extension for Michaelis-Menten analysis
What are
enzymes?
Molecules, usually
proteins, that speed
up the rate of a
reaction by
decreasing the
activation energy
required without
themselves being
altered or used up
Enzyme Class
Example
Oxidoreductase
Firefly Luciferase – oxidizes
luciferin to produce oxyluciferin
and light
Transferase
Hexokinase – transfers a
phosphate group to glucose to
make glucose-6-phosphate
Hydrolase
Cellobiase – breaks down
cellobiose
Lyase
Histidine decarboxylase –
generates histimine from histidine
Isomerase
Glucose-6-Phosphate isomerase –
converts G-6-P to fructose-6phosphate
Ligase
DNA Ligase – covalently bonds two
pieces of DNA
(transfer of electrons)
(group-transfer
reactions)
(hydrolysis reactions)
(double bond
reactions)
(transfers to create a
new isomers)
(forms covalent bonds)
Substrate (S)
How do
enzymes
work?
Energy
considerations
Enzyme
Product (P)
S*
E
N
E
R
G
Y
S*enz
Eact Eact
S
P
REACTION COORDINATE
How do
enzymes
work?
Substrate free in
solution
Substrate binds to a
specific cleft or groove
in the enzyme
Physical
considerations
Activation energy
barrier is overcome and
reaction occurs
Product is released and
enzyme is free to catalyze
another reaction
What are
biofuels?
Fuels that are produced from a
biological source
• Oil – biofuel, but very long
production cycle (millions of
years)
Short cycle Biofuels
• Biodiesel
• Ethanol from starches/sugars
• Cellulosic ethanol
•Butanol
Cellulosic
ethanol
production
A
B
C
D
Cellulose breakdown
1. Heat, acid,
ammonia or
other treatment
2. Enzyme
mixture added
Glucose
Endocellulases
Exocellulases
Cellobiase
Cellobiose
breakdowna closer look
4
1
4
6
5
3
+
Cellobiose + H2O
2
1
2 Glucose
Protocol
Highlights:
Using a
colorimetric
substrate to
track reaction
rate
• Cellobiose and glucose are colorless when
dissolved
• Use of the artificial substrate p-nitrophenyl
glucopyranoside allows the reaction to be
tracked by monitoring the appearance of
yellow color
cellobiose
p-nitrophenyl glucopyranoside
Cellobiase
breakdown of pnitrophenyl
glucopyranoside
+
p-nitrophenyl glucopyranoside + H2O
glucose
+
p-nitrophenol
Basic
conditions
Clear
Yellow
Biofuels
Activity 1
Overview
How can this
enzymatic
reaction be
easily
quantified?
Basic solution (STOP SOLUTION):
- will develop color of any p-nitrophenol
present
- will stop the reaction
• Qualitative - Each reaction time point can
be directly compared to a standard of
known concentration of p-nitrophenol
• Quantitative- The amount of yellow color
in the reaction solution can be quantified
by measuring the absorbance at 410 nm
using a spectrophotometer or microplate
reader.
Biofuel Enzyme Kit
Procedure
Overview
Collaborative approach:
• Each student group
does activity 1
• Student groups do one
activity each from 2-5
• Groups share data
• All groups do activity 6
and share data
Activities:
1. Reaction Rate & Std curve
2. Effect of Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of Substrate
Concentration
6. Bio-prospecting for
Celliobiase
S1
0
0
S2
12.5
0.2
S3
25
0.4
S4
50
0.8
S5
100
1.6
410 nm
Standard Curve
1.8
Absorbance at 410 nm
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Absorbance
Standard
Amount of
p-nitrophenol
(nmol)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Amount of p -nitrophenol (nmol)
120
Standard Curve
1.8
Absorbance at 410 nm
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Amount of p -nitrophenol (nmol)
120
Reaction Rate with Enzyme
Amount of p -nitrophenol
(nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
100
80
60
40
20
0
0
2
4
6
8
10
Time (min)
Initial reaction rate =
Amount of p-nitrophenol
produced (nmol)
Time (min)
Initial reaction rate =
50 nmol - 0 nmol
4 min - 0 min
= 12.5 nmol/min
Activity 2 : Effect of Temp on Reaction Rate
rate p-nitrophenol produced
(nmol/min)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
100
90
80
70
60
50
40
30
20
10
0
Expon.
0
10
20
Temperature (C)
30
40
Initial reaction rate =
Time (min)
•This is the amount of p-nitrophenol produced in
2 minutes
Effect of pH on Initial Reaction Rate
20
Rate of p -nitrophenol
produced (nmol/min)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Amount of p-nitrophenol
produced (nmol)
18
16
14
12
10
8
6
4
2
0
4
5
6
7
pH
8
9
Amount of pnitrophenol formed
(nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
High enzyme
concentration
Low enzyme
concentration
Time (minutes)
1. The initial reaction rate is faster when
there is a higher enzyme concentration
2. Given enough time, the same amount of
product will be formed for both the high
and low enzyme concentration reactions
Amount of pnitrophenol formed
(nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
1.5 mM
substrate
[High]
0.25 mM
substrate
[Low]
Time (minutes)
1. Effect of substrate
concentration on the initial rate
2. Final amount of product
formed with varying substrate
concentrations
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Where can we find things
that break down cellulose?
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Mushrooms – Ecological niche for food
•Mycorrhizal –associated with plant roots
•Porcini
•Chanterelle
•Saprotrophic – decomposers
•Shiitake
•Morel
•Button
•Parasitic – attacks plants
•Honey Mushroom
•Endophytes – grows in plant cells
Activity 6
Protocol
1. Pick a mushroom
2. Add 0.25g of mushroom to
microcentrifuge tube
3. crush with blunted pipette tip
4. Add 500 µl extraction buffer and
continue crushing
5. Spin down extract in microcentrifuge
to separate mushroom particle from
liquid fraction
Activity 6
Protocol
6.
7.
8.
Activity 6
Protocol
SmartSpec™
Plus
Photodiode Array UV-VIS Spectrometer
Measures


Absorbance , %T
Specifications




Range: 200-800 nm
Optical Resolution: ± 2 nm
Light Source: Xenon Flash Lamp
Power: 120 VAC, 60 Hz
Standalone Research Grade Instrument
170-2525EDU
SpectroVis®
Plus
CCD Array VIS-NIR Spectrometer/Fluorometer

Measures Multiple Parameters




Specifications





SVIS-PL
Absorbance , %T
Fluorescence (excitation at 405 or 500 nm)
Emissions Spectra (fiber optic cable required)
Range: 380950 nm
Optical Resolution: ± 2.5 nm
Reporting Interval: 1 nm
Light Source: LED boosted tungsten bulb
Power: USB port
LabQuest or Logger Pro Software required
S1
0
0
S2
12.5
0.2
S3
25
0.4
S4
50
0.8
S5
100
1.6
410 nm
Standard Curve
1.8
Absorbance at 410 nm
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Absorbance
Standard
Amount of
p-nitrophenol
(nmol)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
120
Amount of p -nitrophenol (nmol)
Y = mx + b, solve for X
M = slope
b = y-intercept (can use 0 for ease)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Derive p-nitrophenol
concentrations from Abs data
X = (y-b)/m
Absorbance
Time
#1 – 1 min
#2 – 2 min
#3 – 4 min
#4 – 6 min
#5 – 8 min
#6 - Blank
410 nm
Amount of
p-nitrophenol
(nmol)
125
Add your Data! (general trend line is fine)
P-nitrophenol (nmol)
100
75
50
25
1
2
3
4
Time (minutes)
5
6
7
8
P-nitrophenol (nmol)
1. Std curve / Std
Reaction Rate
2. Effect of
Temperature
3. Effect of pH
4. Effect of Enzyme
Concentration
5. Effect of
Substrate
Concentration
6. Bio-prospecting
for Celliobiase
Time (minutes)
Rate correlates to ecological niche which is
based upon its evolution
Chromosome 16:
PV92 PCR
Informatics
Kit Advantages
• Aligns with new AP Biology Big Ideas 1 & 3
• Inquiry: Extract genomic DNA and amplify
student samples
• Introduce the polymerase chain reaction (PCR)
• Apply PCR to population genetics / evolution
– Hardy-Weinberg analysis
• Directly measure human diversity at the
molecular level
• Compare results to online Alu data; DNA
Learning Center
• Sufficient materials for 8 student workstations
• Complete activity in three 45 minute sessions
Additional
Resources
• Investigating Biology through Inquiry by
Vernier
• Dropbox curriculum folders
– Powerpoints, electronic copies of manuals,
tips/tricks, links to animations/videos, etc
– eMail your CTS
• 20 Questions to Master Inquiry
Bio-Rad
CTS
Contacts
& Vernier
Biology
Specialists
Bio-Rad Curriculum and Training Specialists:
Sherri Andrews, Ph.D. – Eastern U.S.
sherri_andrews@bio-rad.com
Damon Tighe – Western U.S.
damon_tighe@bio-rad.com
Leigh Brown, M.A. – Central U.S.
leigh_brown@bio-rad.com
Vernier:
John Melville
jmelville@vernier.com
Michael Collins
mcollins@vernier.com