2. Lisa Yoneda Mira Mesa High School Teacher Background:
Biology to AP Biology
Biotechnology
Background:
BS Molecular Biology
MA Educational Technology
LSSI alumni (2nd year)
Coordinate UCSD ScienceBridge Kits

3. Laboratory 6: Getting what we need-Protein
LSSI Alum
Lisa Yoneda, Biotechnology Program, Mira Mesa HS

4. Safety 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 Safety Guidelines for lab 6
When using potentially bio-hazardous materials work in a sanitary manner and treat all waste as a potential biohazard
Dispose of pipette tips and all other materials that came in contact with bacteria in the biohazard bag provided
Any item potentially contaminated by bacteria should be treated with 70% ethanol or another acceptable disinfectant

5. NGSS Learning Expectations:
HS-LS1-6: Construct and revise an explanation based on evidence for how carbon, hydrogen, and oxygen from sugar molecules may combine with other elements to form amino acids and/or other large carbon-based molecules
Scientific Practices:
Constructing Explanations and Designing Solutions: Construct an explanation based on valid and reliable evidence obtained from a variety of sources (including students’ own investigations, models, theories, simulations, peer review) and the assumption that theories and laws describing the natural world operate today as they did in the past and will continue to do so in the future
Asking Questions and Defining Problems: Ask questions that arise from examining models or a theory to clarify relationships
Engaging in Argument from Evidence: Make and defend a claim based on evidence about the natural world that reflects both scientific knowledge and student-generated evidence

6. NGSS Disciplinary Core Ideas:
LS1.A: Structure and Function: All cells contain genetic information in the form of DNA molecules. Genes are regions in the DNA that contain the instructions that code for the formation of proteins which carry out most of the work of cells.
LS1.C: Organization for Matter and Energy Flow in Organisms: The sugar molecules thus formed contain carbon, hydrogen, and oxygen. Their hydrocarbon backbones are used to make amino acids and other carbon-based molecules that can be assembled into larger molecules (such as proteins or DNA), which can then be used, for example, to form new cells.

7. Common Core State Standards
English Language Arts/Literacy:
RST : Synthesize information from a range of sources (e.g., texts experiments, simulations) into a coherent understanding of a process, phenomenon, or concept, resolving confliciting information when possible.
WHST : Write arguments focused on discipline-specific content.
WHST : Draw evidence from informational texts to support analysis, reflection, and research

8. 21st Century Core Competencies
Cognitive Domain
Cognitive Processes and Strategies:
Critical Thinking
Problem solving
Analysis
Reasoning / Argumentation
Interpretation
Knowledge:
Oral and Written Communication
Active Listening
Interpersonal Domain: Teamwork and Collaboration
Communication
Collaboration
Teamwork
Interpersonal Skills

9. Lab Prep/Aliquoting Guide (10 Student Groups per Class)
Reagents/Supplies
Aliquot
Storage Temp
Notes
1 flask of E.coli expressing RFP (EC). Alternatively use plate from Lab 5 to inoculate 100ml LB/Amp/Ara. PLEASE REQUEST Culture or LB and Amp/Ara when making reservation on LARS. You will need ~20ml/class.
Aliquot 1ml/group to start. Students will centrifuge then remove liquid and you will aliquot 1 additional ml/group.
Fridge (4o)
Please return flask
10 Resin packed columns stored in Column Equilibration Buffer (CEB)
N/A RT
Please return columns
10 tubes with 15 mls of Elution Buffer/ kit (EB)
50 mls extra/kit
Please return ALL tubes
10 tubes with 15 mls of Wash Buffer/kit (WB)
10 tubes with 15 mls of Column Equilibration Buffer/kit (CEB)
10 tubes with 5 mls of Binding Buffer/kit (BB)
20 mls extra/kit
1.6 ml of Lysis Buff/class (LyB)
160ul/group
Equipment/Supplies:
10 Student boxes with the following:
1 p20 micropipette microfuge tube rack
1 p200 micropipette bag of microfuge tubes
1 box of refillable tips (2 ul-200 ul) ice bucket
Laminated pipetting practice card waste bucket
4 Mini centrifuges
1 Tabletop centrifuge to spin lysed samples at 10,000rpm for 5 minutes
10 Ring stands with clamps
10 Boxes of p1000 tips
10 p1000 pipets – please note that these get broken frequently. Please do not let students adjust above 1000ul or below 200ul. They are EASILY broken.
Optional: Shaker for preparing the RFP culture.

11. Warm Up Why is your transformed bacteria red? What did you do?
What did the bacteria do?

12. Transformation

13. Transcription & Translation
Where is rfp?
Is rfp the only protein made by the cell?

14. Why purify a protein?
The protein is in living cells and mixed in with other proteins, over 1, 000 proteins could be in one cell
Pharmaceutical companies want one purified protein to sell as a medicine.
other proteins may interfere with the medicine or body chemistry

15. How to Purify a Protein Procedure Purpose increase concentration
Centrifuge - transformed liquid culture
Lyse cells- overnight incubation
Centrifuge – lysed cells
Column – Purification of rfp
increase concentration
Release cell proteins (including rfp)
Separate large cell pieces from released proteins
Separate rfp from other cell proteins

16. Where’s your protein? How will you know where your protein is?
Create a flowchart for each step of the procedure
Record where rfp should be (prelab)
As you complete- check location of rfp!

17. Part A (Day 1)
Step 1: Centrifuge
Step 2: Lyse cells

18. Outline Part A Protocol (prelab)
Notes/Changes
Flow chart of each main step
Draw with labels
Be sure to note where the rfp should be for each step
Enough details that can use your notes to do the lab
Amounts
Time
Name of solutions

19. Step 1: Centrifuge
A) 1 mL rfp cell culture
What will happen when you centrifuge your cell culture?
B) Centrifuge
5 min
(high speed)
What will happen when you centrifuge your cell culture?

20. Step 1: Centrifuge What is in each of the separated parts?
A) 1 mL rfp cell culture
B) Centrifuge
5 min
(high speed)
What is in each of the separated parts?
Supernatant – broth / waste
Pellet – rfp inside cells

21. Step 1: Centrifuge
C) Discard supernatant (Keep pipet tip away from pellet- pipet slow)
A) 1 mL rfp cell culture
B) Centrifuge
5 min
(high speed)
Supernatant – broth / waste
Pellet – rfp inside cells

22. Step 1: Centrifuge
C) Discard supernatant (Keep pipet tip away from pellet- pipet slow)
A) 1 mL rfp cell culture
B) Centrifuge
5 min
(high speed)
Supernatant – broth / waste
Pellet – rfp inside cells
D) Repeat steps A-C in the SAME tube for bigger pellet – more rfp

23. Step 2: Lyse Cells
What will happen when we lyse the cells?

24. Step 2: Lyse Cells Flow Chart
A) Resuspend with 150 uL EB
Where is rfp?
Cell Pellet (step 1)

25. Step 2: Lyse Cells Flow Chart
B) Add 150 uL LB
A) Resuspend with 150 uL EB
C) Overnight incubation
What does LB do?
Resuspended cells
Cell Pellet (step 1)
Bacterial proteins
rfp

26. Part A (Day 1) Overview Step 1: Centrifuge
1 mL of rfp to microfuge tube
Centrifuge at high speed for 5 min
Be sure to balance tubes
Remove supernatant without disturbing pellet
Repeat in the SAME microfuge tube!
End product: 1 microfuge tube with red pellet

27. Part A (Day 1) Overview Step 2: Lyse cells
Add 150 µL EB (buffer) to cell pellet
Re-suspend cells
No clumps- use spiral notebook
Increase surface area for lysozyme
Add 150 µL LB (lysozyme) to cells and mix- use spiral notebook
Incubate Overnight (room temp)
Can freeze if longer

28. Protein Purification Procedure Where is rfp?
Centrifuge - transformed liquid culture
Lyse cells- overnight incubation
Cytoplasm inside the cell
Liquid portion (cells cut open)

29. Use your flowgram to complete Lab 6 Part A
Complete Part A (Day 1)
Use your flowgram to complete Lab 6 Part A
Step 1: Centrifuge
Step 2: Lyse cells

30. Part A (Day 1) Teacher Prep Notes
Can pre aliquot 1000 uL rfp into student tubes (1/group)
If pressed for time can do 2/group and then have them combine (add EB to 1 pellet-mix and transfer into second pelleted tube)
Pre-label collection rack (1/period)
If have sensitive centrifuge recommend having students use a balance for 2nd spin