Download Space Food Safety Lab Classroom Slides

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miniPCR
Space Food Safety Lab
Mars Colony at risk!
TM
Release v4.0: November 2016
© Copyright by Amplyus LLC, all rights reserved
2021: A Food Safety Crisis in Space
Houston, we have a
(food safety) problem!
 E.coli contamination in
International Space Station
 Food in transit to Mars
 Find source of E.coli O157:H7
 Use DNA analysis
 PCR
 Restriction digest
 Gel electrophoresis
© Copyright by Amplyus LLC, all rights reserved
1
The International Space Station
offers a multicultural food experience
Source: Wikipedia
2
Food Safety is a major public
health concern on Earth
Source: NBC News
3
Biotechnology can help us contain the outbreak
Micropipetting
Prepare
samples
PCR
Amplify bacterial
genes
Frist class period
Restriction
digest
Identify
E. coli strains
Gel
electrophoresis
Visualize
results
Second class period
4
Reminder: DNA’s unique structure
DNA: a double helix...
...held together by base complementarity
Source: US National Library of Medicine, NIH, Thinkquest
5
Step 1
Micropipetting
Prepare
samples
6
Step 1: Micropipetting – Basic practice
1. Identify volume range
2. Adjust volume
3. Press plunger to FIRST STOP
4. Press plunger to SECOND STOP
7
Let’s transfer real liquids: 20, 10, and 5µl
1. Adjust volume
2. Get a tip
3. Press plunger to FIRST STOP
4. Collect liquid. Release plunger
5. Transfer liquid
6. Press plunger to SECOND STOP
7. Eject tip
8
Step 2: Amplify target genes
Micropipetting
Prepare
samples
PCR
Amplify bacterial
genes
9
PCR applications have transformed our world
Molecular
diagnostics
Text
Consumer
genomics
Personalized
medicine
Text
Food and
agriculture
Text
PCR
Text
Text
Human
evolution
Text
Forensics
10
Use PCR to find (and copy) the target gene
Single molecule
Target E. coli
gene
~1B copies
11
Get samples ready to solve this Space Food Mystery
ISS SAMPLES
Sample A: Sushi
A
Space Sushi
aboard ISS
Sample B: Burgers
B
Control P
Controls
P
Pathogenic
E.coli DNA
Space Burgers
aboard ISS
Control NP
NP
Non-pathogenic
E.coli DNA
12
What goes in a PCR experiment
1. Template DNA to be amplified
2. Pair of DNA primers
Taq
3. DNA polymerase
FWD primer
REV primer
4. dNTPs
G
5. Buffer to maintain pH
and provide Mg2+
A
A
C TG
G
TA
C
C
13
Prepare 4 PCR tubes (200 µl)
1
15 µL
2X EZ PCR Master Mix
2
10 µL
3X Primer mix
3
5 µL
Template DNA (Samples A, B, P, NP)
Space
Sushi
Space
Burgers
Pathogenic
Control
Nonpathogenic
control
A
B
P
NP
Add group name to side wall of tube
14
Programming PCR parameters
Initial denaturation:
94°C
30 seconds
Denaturation:
Annealing
Extension
94°C
57°C
72°C
5 seconds
5 seconds
5 seconds
x25 cycles (if prioritizing speed) OR
x30 cycles (for stronger bands)
Final extension
72°C
30 seconds
15
Foodborne pathogens: identifying the culprit bacteria
• Most Escherichia coli are
harmless living in our gut
• Pathogenic strains can be
serotyped by O/H antigens
• 0157:H7 strains can cause
severe hemorrhagic
diarrhea
• Complications can cause
kidney damage, even death
• Bacterial culture, antigen
detection are slow and
inefficient  USE PCR
16
Amplify bacterial DNA exponentially
17
Polymerase Chain Reaction (PCR)
Complex DNA
sample
Region of
interest
Amplified DNA
(Billions of copies)

Sequencing

Genetic risk

Pathogen detection

Drug development

Crop modification

Forensic analysis

Etc.
Applications
A process that identifies and copies (amplifies)
a specific piece of DNA in a biological sample
18
How PCR works: 3 steps to copy DNA
94°C
1
Denaturation
2
50-60°C
Primer 1
Annealing
Primer 2
72°C
3
Extension
Taq DNA
polymerase
dNTPs
19
How PCR works: repeat the cycle
denatured DNA
Single molecule
DNA + primers
DNA + copy
94° C
72° C
~1B copies
50-60° C
Denaturation
Annealing
Extension
Repeat x ~25-30 cycles
20
What’s happening at each step?
?
primers
Taq
Single molecule
~1B copies
How many DNA molecules are in your tube now?
21
Monitoring DNA amplification
What is happening to DNA molecules at each PCR step?
• Denaturation
• Annealing
• Extension
What is unique about Taq DNA polymerase?
• What temperature is optimal for most enzymes?
How many molecules of DNA will we have with each PCR cycle?
• And at the end of the PCR?
22
First PCR in Space:
Genes in Space experiment designed by a high school student!
www.GenesinSpace.org
23
Quiz: Which of these are NOT characteristics of PCR primers?
 A. Short synthetic oligonucleotide
 B. Typically 18-25 bases in length
 C. Double stranded DNA
 D. Unique homology to the DNA template
 E. Sequence with ~50% G:C content
24
Second class period:
Restriction Digest and Electrophoresis
Micropipetting
Prepare
samples
PCR
Amplify bacterial
genes
Restriction
digest
Identify
E.coli strains
Gel
electrophoresis
Visualize
results
NEXT STEP
25
A single nucleotide difference can solve this mystery
Target gene
fliC
(non-pathogenic
E.coli strain)
XmnI site
Target gene
(pathogenic E.coli
O157:H7 strain)
fliC
SNP
Genetic difference between E.coli strains
• Single nucleotide polymorphism (SNP) in fliC gene
• Creates unique restriction site in pathogenic E.coli
26
Set up restriction digest (XmnI)
PCR
product
A
15 µL
+
1 µL
Restriction
digest
(incubate 15
min at 37°C)
B
P
NP
Restriction enzyme
(XmnI restriction endonuclease)
AX
BX
PX
NPX
27
Incubate 15 minutes at 37°C
Use miniPCR
machine as
heat block
28
Restriction enzymes: molecular scissors
Cuts DNA at specific nucleotide
sequences known as restriction sites
• Found in bacteria
• Defense mechanism against
invading viruses
• Used as tools in the lab
Source: Wikipedia
29
Experimental overview
Set-up
PCR
Restriction
digest
Electrophoresis
Gel
& visualization
electrophoresis
fliC
400bp
400bp
400bp
150+250bp
Non-pathogenic
XmnI site
fliC
O157:H7
30
Step 4: Gel electrophoresis
Micropipetting
Prepare
samples
PCR
Amplify bacterial
genes
Restriction
digest
Identify
strains
Gel
electrophoresis
Visualize
results
31
Gel electrophoresis
1. Pour an agarose gel
3. Electrophoresis
2. Load the PCR products
4. Visualization in a transilluminator
- Pole
e+ Pole
32
Integrated gel
electrophoresis and
visualization system
33
Cast 2% agarose gels
►
0.4 g agarose
►
20 ml 1X TBE
buffer
►
Heat for
20-30 sec
►
Add 2 µl
GreenView™
Plus stain
►
Pour into
casting tray
with comb
9 lanes needed per lab group
34
Load gel as follows:
Don’t pierce bottom!
12 µl per lane
Ladder
A B P NP Ax Bx Px NPx
35
Predict results
Ladder
A
B
P
NP Ax Bx Px NP
x
36
Final Questions – Where did the contamination come from?
How did the investigation turn out?
Which food might be the source of the outbreak?
What’s the importance of running controls?
What was the most surprising thing you learned?
37
www.genesinspace.org
38
Thank you - We hope you enjoyed the lab!
www.miniPCR.com
[email protected]
Facebook.com/miniPCR
39
Open inquiry - variants of this lab
Variant 1: Open inquiry
Testing more than 2 foods
• Most lab groups receive multiple NP DNA samples
• Selected groups receive P DNA (~2-3 groups per class)
• Students collaborate and exchange data to establish source of outbreak
• Some groups can receive mixed A + B DNA samples (multiple strains in sample)
Variant 2: Split roles
Food safety inspectors vs. Control Reference Lab
• Food Safety Inspectors: Groups process DNA Samples A and B only
• Control Reference Lab: Groups process controls (P and NP) only
• Students must collaborate and match data to establish source of the outbreak
40
Additional resources
Outbreak Detection Since Jack in the Box: A Public Health Evolution
http://www.foodsafetynews.com/2013/02/outbreak-detection-since-jack-in-the-boxa-public-health-evolution/#.VNre1J3F-Sp
CDC PulseNet Home
http://www.cdc.gov/pulsenet/
Centers for Disease Control: E.coli outbreaks
http://www.cdc.gov/ecoli/outbreaks.html
Popular books around E.coli O157:H7 outbreaks
• Toxin (by Robin Cook):
http://en.Wikipedia.org/wiki/Toxin_%28novel%29
• Poisoned (by Jeff Benedict):
http://www.amazon.com/Poisoned-Deadly-Outbreak-ChangedAmericans/dp/098495435X
41
Foodborne Illness Outbreaks:
Major Public Health Concern
• 48M Americans get sick each year
• 128,000 are hospitalized
• 3,000 die
Source:
http://www.cdc.gov/ecoli/general/index.html#what_shiga
and http://www.cdc.gov/ecoli/outbreaks.html
Foodborne illnesses cost the
economy more than $15.6 billion / yr
42
The complete biotech toolkit: DNA Discovery System™
Samples
PCR
Electrophoresis
Visualization
43