laboratory 5: transforming bacteria with ligation products miracosta college: amgen site in...
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LABORATORY 5: TRANSFORMING BACTERIA WITH LIGATION PRODUCTSMiraCosta College: Amgen Site in Oceanside, CA Dr. Annie Holland, Professor of Biotechnology
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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 GuidelinesReagents/Supplies Aliquot Storage Temp Notes
110uL Competent Cells--10 tubes/ class (CC or Cells)
Aliquoted for you
-20o for 2 weeks Keep LB/Amp/Ara plate with pink colonies stored at (4o) to use for inoculating culture for lab 6350 ul LB broth --10 tubes/ class
(LB)Aliquoted for you
4o
11 LB plates/class (1 blue line) N/A 4o
11 LB/Amp plates/ class (2 blue lines)
N/A 4o
11 LB/Amp/Ara plates / class (3 blue lines)
N/A 4o
Equipment/Supplies10 Student boxes with the following:
1 p20 micropipette 1 microfuge rack1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes1 waste and 1 ice bucket 1 box of refillable tips (2 ul-200 ul)
4 Mini centrifuges1 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)
Emphasize!!!
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Making E. coli Cells “Competent” for DNA Uptake (“Transformation”)
Adhesion zone
Calcium ions
Lipid bilayer (inner)
Peptidoglycan layer
Lipid bilayer (outer)
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Introducing New Plasmid Into Host E. coli Cells (“Heat Shock” or “Transformation”)
Recombinant Plasmids
Amp Sensitive Competent Cells (CC, provided)
+
Step 2: Ice, then 42°C,
then ice
Step 1: Mix
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What Happens During Heat Shock
Calcium ions
pARA-R
Adhesion zone
Lipid bilayer (inner)
Peptidoglycan layer
Lipid bilayer (outer)
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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)
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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)
– AmGen Lab 5-Intro Card Sort 2 Activity (Central Dogma)
– Learning Goal: Describe the role of transformation in the gene cloning process
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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 5Session 2Flowchart
Student Guide pages B93-94:
Stop at step 7 Stop here for
more discussion
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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
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Lab 5Session 2Flowchart
Student Guide page B94:
Resume at step 8
continue
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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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Inoculating Plates
Repeat with P+
Label plates Inoculate LB and LB/amp Plates with P-
Spread
Continue student guide page B95 step 13:
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Incubating Plates
End of session 2 Session 3
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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
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
Learning Goal #2: Explain the purpose of each control in the transformation experiment
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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/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