LABORATORY 5: TRANSFORMING BACTERIA WITH LIGATION PRODUCTS MiraCosta College: Amgen Site in Oceanside, CA Dr. Annie Holland, Professor of Biotechnology

LABORATORY 5: TRANSFORMING BACTERIA WITH LIGATION PRODUCTSMiraCosta College: Amgen Site in Oceanside, CA Dr. Annie Holland, Professor of Biotechnology

www.amgenbiotechexperience.com

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)


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


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


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.)


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


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)

http://www.matsc.org/Amgen%20content/Amgen%20SOPs/SOP%20Operation%20of%20the%20ISOtemp%20AquaBath%20Water%20Bath.pdf


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


Recombinant Construct (Labs 2-4)

Emphasize!!!


Making E. coli Cells “Competent” for DNA Uptake (“Transformation”)

Adhesion zone

Calcium ions

Lipid bilayer (inner)

Peptidoglycan layer

Lipid bilayer (outer)


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


What Happens During Heat Shock

Calcium ions

pARA-R

Adhesion zone

Lipid bilayer (inner)

Peptidoglycan layer

Lipid bilayer (outer)


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


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

http://www.dnaftb.org/34/animation.html





– Alternatively, video explaining theory behind heat shock transformation play video (2 minutes 36 sec)


https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/transforming-bacteria






– AmGen Lab 5-Intro Card Sort 2 Activity (Central Dogma)








– AmGen Lab 5-Intro Card Sort 2 Activity (Central Dogma)

– Learning Goal: Describe the role of transformation in the gene cloning process




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


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

https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/transforming-competent-cells


Lab 5Session 2Flowchart

Student Guide pages B93-94:

Stop at step 7 Stop here for

more discussion




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



Lab 5Session 2Flowchart

Student Guide page B94:

Resume at step 8

continue


Lab 5 Flowchart continued

Stop here for plating video(step 12 student guide pg B95)






learning goal

– Video demonstrating plating cells play video (4 minutes 56 seconds)


https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/plating






learning goal

– Video demonstrating plating cells play video (4 minutes 56 seconds)

– Learning Goal: Explain the purpose of each control in the transformation experiment




Inoculating Plates

Repeat with P+

Label plates Inoculate LB and LB/amp Plates with P-

Spread

Continue student guide page B95 step 13:


Incubating Plates

End of session 2 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



Lab 5 Suggested Sequence of Activities

• Session 3: Collect and analyze data, clean up (Student Guide page B97 steps 22-23)


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


Expected Growth (UV light)

P+P-

P+P-

P+



• 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







– Can Play TIC-BAC-TOE







– Can Play TIC-BAC-TOE– Demonstrate overnight culture if doing lab 6 (video useful

for teacher refresher play video)

https://www.amgenbiotechexperience.com/curriculum/curriculum-resources/picking-colony-plate


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


• Plating (performing lab 5 part 2: 4 min 56 sec), 2012 Amgen Foundation/WGBH Educational Foundation Play Video


• Picking a Colony From a Plate (to set up lab 6, 1 min 23 sec), 2012 Amgen Foundation/WGBH Educational Foundation Play Video







Documents

LABORATORY 5: TRANSFORMING BACTERIA WITH LIGATION PRODUCTS MiraCosta College: Amgen Site in Oceanside, CA Dr. Annie Holland, Professor of Biotechnology