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)

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 Guidelines Reagents/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 6 350 ul LB broth --10 tubes/ class (LB) 4o 11 LB plates/class (1 blue line) N/A 11 LB/Amp plates/ class (2 blue lines) 11 LB/Amp/Ara plates / class (3 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

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)

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”) Lipid bilayer (inner) Peptidoglycan layer Lipid bilayer (outer) Adhesion zone Calcium ions

Amp Sensitive Competent Cells (CC, provided) Introducing New Plasmid Into Host E. coli Cells (“Heat Shock” or “Transformation”) Step 1: Mix Recombinant Plasmids Step 2: Ice, then 42°C, then ice + Amp Sensitive Competent Cells (CC, provided)

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)

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

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)

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)

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

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

Lab 5 Session 2 Flowchart Student Guide pages B93-94: Stop at step 7 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

Lab 5 Session 2 Flowchart Student Guide page B94: Resume at step 8 continue

Lab 5 Flowchart continued Stop here for plating video (step 12 student guide pg B95)

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)

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

Inoculate LB and LB/amp Plates with P- Inoculating Plates Continue student guide page B95 step 13: Label plates Inoculate LB and LB/amp Plates with P- Repeat with P+ Spread

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 LB LB/amp LB/amp/ara P- plates Learning Goal #2: Explain the purpose of each control in the transformation experiment No growth LB LB/amp

Expected Growth (UV light)

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

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

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)

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