LABORATORY 5: TRANSFORMING
BACTERIA WITH LIGATION PRODUCTS
MiraCosta College: Amgen Site in Oceanside, CA
Dr. Annie Holland, Professor of Biotechnology
Lab 5: Transformation of E. coli
with p-ARA-R
Objective:
• Transform E. coli with pARA-R containing the
rfp gene of interest
• Culture transformed cells for use in Lab 6 as a
source of red fluorescent protein for
purification (optional)
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Overview
•
•
•
•
Safety guidelines
Materials: Advance Teacher Prep, Aliquoting
Learning Goals
Suggested Lab 5 activities and class sessions
– Session 1: theory of transformation, preparation for
lab 5 performance
– Session 2: Lab 5 – transform competent E. coli cells,
plate
– Session 3: examine results of plating after overnight
growth
• Useful Links and Resources
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General Lab Safety Guidelines
•
•
•
•
Use laboratory coats, safety glasses and gloves as appropriate
Avoid restrictive clothing and open-toed shoes
No eating or drinking in the lab
Make sure that students are familiar with the operating
instructions and safety precautions before they use any of the lab
equipment
• Check all MSDS (Material Safety Data Sheets) for all chemicals and
reagents in the lab before preparing and running the lab
• Wash hands at the conclusion of the lab
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Lab 5-Specific Safety Guidelines
• When using potentially bio-hazardous materials work in a sanitary
manner, and treat all waste as a potential biohazard
• Dispose of petri dishes, pipette tips and all other materials that
came in contact with bacteria in the biohazard bag provided
(return bag with kit for decontamination)
• Any surface, item or liquid potentially contaminated by bacteria
should be treated with 70% ethanol or another acceptable
disinfectant (20% bleach, Lysol, etc.)
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Lab Prep & Aliquoting Guidelines
Reagents/Supplies
Aliquot
Storage Temp
Notes
110uL Competent Cells--10 tubes/ Aliquoted
-20o for 2 weeks Keep LB/Amp/Ara plate with pink
class (CC or Cells)
for you
colonies stored at (4o) to use for
350 ul LB broth --10 tubes/ class
Aliquoted
4o
inoculating culture for lab 6
(LB)
for you
11 LB plates/class (1 blue line)
N/A
4o
11 LB/Amp plates/ class (2 blue
N/A
4o
lines)
11 LB/Amp/Ara plates / class (3
N/A
4o
blue lines)
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 microfuge rack
1 p200 micropipette
1 bag of microfuge tubes
1 p1000 micropipette
1 bag of microfuge tubes
1 waste and 1 ice bucket
1 box of refillable tips (2 ul-200 ul)
4 Mini centrifuges
1 Water bath
1 Incubator
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Equipment for Lab 5, Session 2
• Water bath should be stabilized at 42°C before classes begin (for
administering heat shock)
• Incubator should be stabilized at 37°C before classes begin (for
incubating plates after performance of lab 5)
On MATSC.org:
• Operation of the ISOtemp AquaBath Water Bath Rev 01 (PDF)
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Learning Goals for Lab 5
• Describe the role of transformation in the
gene cloning process
• Explain the purpose of each control in the
transformation experiment
• Explain how the information encoded in a
gene is expressed as a trait
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Recombinant Construct (Labs 2-4)
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Making E. coli Cells “Competent” for DNA
Uptake (“Transformation”)
Adhesion
zone
Calcium
ions
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Lipid bilayer (inner)
Peptidoglycan layer
Lipid bilayer (outer)
Introducing New Plasmid Into Host E. coli
Cells (“Heat Shock” or “Transformation”)
Step 1:
Mix
Recombinant
Plasmids
+
Amp Sensitive
Competent Cells
(CC, provided)
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Step 2: Ice,
then 42°C,
then ice
What Happens During Heat Shock
Lipid bilayer
(inner)
Peptidoglycan
layer
Adhesion
zone
Lipid bilayer
(outer)
Calcium ions
pARA-R
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Lab 5 Suggested Sequence of Activities
(Overview)
• Session 1: Active reading and class discussion
– Questions from lab guide
– Video explaining theory behind heat shock
transformation (2 minutes 36 seconds)
– Central Dogma of Biology (card-sorting activity)
• Session 2: Perform Lab 5 “Transforming Bacteria With
Recombinant Plasmids”
– Activity contains two 15-minute incubations
– Two videos demonstrate necessary techniques
• Session 3: Collect and analyze data
– Instructor demonstrates setup for Lab 6
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Lab 5 Suggested Sequence of Activities
• Session 1: Active reading and class discussion
– “What Do You Already Know”, “Consider”, and
“Before the Lab” Questions (Student Guide pages
B85, B86-87, and B91-92, respectively)
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Lab 5 Suggested Sequence of Activities
• Session 1: Active reading and class discussion
– “What Do You Already Know”, “Consider”, and
“Before the Lab” Questions (Student Guide pages
B85, B86-87, and B91-92, respectively)
– Big picture animation and historical perspective
play video
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Lab 5 Suggested Sequence of Activities
• Session 1: Active reading and class discussion
– “What Do You Already Know”, “Consider”, and
“Before the Lab” Questions (Student Guide pages
B85, B86-87, and B91-92, respectively)
– Big picture animation and historical perspective
play video
– Alternatively, video explaining theory behind heat
shock transformation play video (2 minutes 36
sec)
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 1: Active reading and class discussion
– “What Do You Already Know”, “Consider”, and
“Before the Lab” Questions (Student Guide pages
B85, B86-87, and B91-92, respectively)
– Big picture animation and historical perspective
play video
– Alternatively, video explaining theory behind heat
shock transformation play video (2 minutes 36
sec)
– AmGen Lab 5-Intro Card Sort 2 Activity (Central
Dogma)
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 1: Active reading and class discussion
– “What Do You Already Know”, “Consider”, and
“Before the Lab” Questions (Student Guide pages
B85, B86-87, and B91-92, respectively)
– Big picture animation and historical perspective
play video
– Alternatively, video explaining theory behind heat
shock transformation play video (2 minutes 36
sec)
– AmGen Lab 5-Intro Card Sort 2 Activity (Central
Dogma)
– Learning Goal: Describe the role of transformation
in the gene cloning process
www.amgenbiotechexperience.com
Session 2: Student Workflow Overview
• Keep everything on crushed ice
• Label tubes and aliquot cells; add plasmid
to P+
• Label plates; review aseptic technique and
spreading technique
• Heat shock; recover (add LB)
• Inoculate/spread plates; incubate (upside
down) 37C overnight
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Lab 5 Suggested Sequence of Activities
• Session 2: Lab 5 “Transforming Bacteria With
Recombinant Plasmids”
– Video demonstrating mixing cells with plasmid play video
2 minutes 52 seconds
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Lab 5
Session 2
Flowchart
Student
Guide
pages
B93-94:
Stop at step
7
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Stop here for
more discussion
Lab 5 Suggested Sequence of Activities
• Session 2: Lab 5 “Transforming Bacteria With
Recombinant Plasmids”
– Video demonstrating mixing cells with plasmid play video
2 minutes 52 seconds
– Debrief “Before the Lab” Questions while cells are on ice;
“Stop and Think” Questions (Student Guide page B95)
– “Before the Lab” and “Stop and Think” questions address second
learning goal
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Lab 5
Session 2
Flowchart
Student
Guide
page B94:
Resume
at step 8
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continue
Lab 5 Flowchart continued
Stop here for plating video
(step 12 student guide pg B95)
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Lab 5 Suggested Sequence of Activities
• Session 2: Lab 5 “Transforming Bacteria With
Recombinant Plasmids”
– Video demonstrating mixing cells with plasmid play video
2 minutes 52 seconds
– Debrief “Before the Lab” Questions while cells are on ice;
“Stop and Think” Questions (Student Guide page B95)
– “Before the Lab” and “Stop and Think” questions address second
learning goal
– Video demonstrating plating cells play video (4 minutes
56 seconds)
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 2: Lab 5 “Transforming Bacteria With
Recombinant Plasmids”
– Video demonstrating mixing cells with plasmid play video
2 minutes 52 seconds
– Debrief “Before the Lab” Questions while cells are on ice;
“Stop and Think” Questions (Student Guide page B95)
– “Before the Lab” and “Stop and Think” questions address second
learning goal
– Video demonstrating plating cells play video (4 minutes
56 seconds)
– Learning Goal: Explain the purpose of each control in the
transformation experiment
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Continue student
guide page B95
step 13:
Inoculating Plates
Label plates
Inoculate LB and LB/amp
Plates with P-
Repeat
with P+
Spread
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Incubating Plates
End of session 2
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Session 3
Session 2: Lab 5 Teacher Prep and Tips
• In advance:
– Equilibrate water bath (42°C) and incubator (37°C)
– Competent cells and LB are already aliquoted for
each group, keep competent cells frozen until lab
• Day of lab:
– Use crushed ice (get from athletic trainer, or use food
processor/snow cone machine/etc.)
– Thaw competent cells (CC) on ice immediately
before class; have students bring ice cup with
chilled tubes to you to obtain CC
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Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page B97 steps 22-23)
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Expected Growth (visible light)
P+ plates
LB
LB/amp
LB/amp/ara
No growth
Learning Goal
#2: Explain the
purpose of each
control in the
transformation
experiment
P- plates
LB
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LB/amp
Expected Growth (UV light)
P-
P-
P+
P+
P+
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Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page B97 steps 22-23)
– Chapter 5 Questions (Student Guide page B98)
– Question 4 addresses first learning goal
– Question 3 addresses second learning goal
– Questions 6 and 7 address third learning goal
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Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page B97 steps 22-23)
– Chapter 5 Questions (Student Guide page B98)
– Question 4 addresses first learning goal
– Question 3 addresses second learning goal
– Questions 5 and 6 address third learning goal
– Can Play TIC-BAC-TOE
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Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page B97 steps 22-23)
– Chapter 5 Questions (Student Guide page B98)
– Question 4 addresses first learning goal
– Question 3 addresses second learning goal
– Questions 5 and 6 address third learning goal
– Can Play TIC-BAC-TOE
– Demonstrate overnight culture if doing lab 6 (video useful
for teacher refresher play video)
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Support Videos – Links and Info
• Transforming Bacteria (theory of how transformation works, 2 min 36
sec), 2011 WGBH Educational Foundation Play Video
https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/transforming-bacteria
• Transforming Competent Cells (performing lab 5 part 1: how to mix
reaction tubes for lab 5, 2 min 52 sec), 2012 Amgen Foundation/WGBH
Educational Foundation Play Video
https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/transformingcompetent-cells
• Plating (performing lab 5 part 2: 4 min 56 sec), 2012 Amgen
Foundation/WGBH Educational Foundation Play Video
https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/plating
• Picking a Colony From a Plate (to set up lab 6, 1 min 23 sec), 2012
Amgen Foundation/WGBH Educational Foundation Play Video
https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/picking-colony-plate
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