Transcript Laboratory 5: Transforming bacteria with ligation products

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)
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
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.)
• Emphasize to students that once a glove is on, assume it is
contaminated– no touching face, phone, door handle, etc. with a
gloved hand!
www.amgenbiotechexperience.com
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
Crushed Ice
1 Water bath
1 Incubator
www.amgenbiotechexperience.com
Equipment for Lab 5, Session 2
• Water bath should be stabilized at 42°C before classes begin (for
administering heat shock) (Turn on first thing on morning, or night
before)
• Incubator should be stabilized at 37°C before classes begin (for
incubating plates after performance of lab 5) (Turn on first thing
in morning, or night before)
– Incubator just plugs in, then set dial to 37 degrees. It comes with a
thermometer inside it- use this reading to fine-tune the dial after you get
close to 37 degrees.
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
Recombinant Construct (Labs 2-4)
www.amgenbiotechexperience.com
Making E. coli Cells “Competent” for DNA
Uptake (“Transformation”)
Adhesion
zone
Calcium
ions
www.amgenbiotechexperience.com
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)
www.amgenbiotechexperience.com
Step 2: Ice,
then 42°C,
then ice
What Happens During Heat Shock (RARE)
DNA
Lipid bilayer
(inner)
Peptidoglycan
layer
Adhesion
zone
Lipid bilayer
(outer)
Calcium ions
pARA-R
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
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
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
85, 86-87, and 91-92, respectively)
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
85, 86-87, and 91-92, respectively)
– Big picture animation and historical perspective
view animation
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
85, 86-87, and 91-92, respectively)
– Big picture animation and historical perspective
view animation
– 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
85, 86-87, and 91-92, respectively)
– Big picture animation and historical perspective
view animation
– 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
Central Dogma Sort Amgen Lab 5 Introduction
Directions: Match and sequence the central dogma of biology.
DNA
Card Sort
Activity:
Central
Dogma
transcription
protein
translation
RNA
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
85, 86-87, and 91-92, 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 wet ice
(slurry of ice and water)
• 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
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
www.amgenbiotechexperience.com
Lab 5
Session 2
Flowchart
Student
Guide
pages
93-94:
Stop at step
7
www.amgenbiotechexperience.com
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 pages 91-92,
and page 95)
– “Before the Lab” and “Stop and Think” questions address second
learning goal
www.amgenbiotechexperience.com
Lab 5
Session 2
Flowchart
Student
Guide
page 9495:
Resume
at step 8
www.amgenbiotechexperience.com
continue
Lab 5 Flowchart continued
Stop here for plating video
(step 12 student guide pg 95)
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 91-92,
95)
– “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 91-5)
– “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
www.amgenbiotechexperience.com
Predictions
Worksheet
(RM5)
www.amgenbiotechexperience.com
Continue student
guide page 95 step
13:
Inoculating Plates
Label plates
Inoculate LB and LB/amp
Plates with P-
Repeat
with P+
Spread
www.amgenbiotechexperience.com
Incubating Plates
End of session 2
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page 97 steps 22-23)
www.amgenbiotechexperience.com
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
www.amgenbiotechexperience.com
LB/amp
Expected Growth (UV light)
P-
P-
P+
P+
P+
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page 97 steps 22-23)
– Chapter 5 Questions (Student Guide page 98)
– Question 4 addresses first learning goal
– Question 3 addresses second learning goal
– Questions 5 and 6 address third learning goal
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page 97 steps 22-23)
– Chapter 5 Questions (Student Guide page 98)
– 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
www.amgenbiotechexperience.com
Lab 5 Suggested Sequence of Activities
• Session 3: Collect and analyze data, clean up (Student
Guide page 97 steps 22-23)
– Chapter 5 Questions (Student Guide page 98)
– 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)
www.amgenbiotechexperience.com
Support Videos – Links and Info
• Cloning Pic Picture Animation, Historical Perspective Show Animation
http://www.dnaftb.org/34/animation.html
• 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/transforming-competent-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
www.amgenbiotechexperience.com