adapting labs for inquiry
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Adapting labs for inquiry. EDS 198 . Agenda. Lab Review and Analysis of Results Adapting Lab for Inquiry Protein Purification Lab Background. Review. What was the purpose of the lab last week? How did we accomplish this?. - PowerPoint PPT PresentationTRANSCRIPT
EDS 198 ADAPTING LABS FOR INQUIRY
Lab Review and Analysis of ResultsAdapting Lab for InquiryProtein Purification Lab Background
AGENDA
What was the purpose of the lab last week?
How did we accomplish this?
REVIEW
The purpose of the lab was to change the genotype of e. coli to change it’s phenotype.
REVIEW: HOW WE MAKE E.COLI GLOW
Bacteria now express cloned fluorescent protein…
Allow bacteria to grow for 1-3 days
Plasmid Uptake of foreign DNA, often a circular plasmid
DNA RNA Protein
REVIEW: 3 AGAR PLATES
LB/No Amp - LB/AMP +
LB/AMP -
What was the purpose of growing bacteria on each plate?
On the post lab section of your protocol, draw out what you expect to see growing on each plate.
ANALYZE: LET’S SEE YOUR PLATES
Demo using a black light
Questions for you and your students to think about:
What do you observe?
Was it what you expected? Why or why not?
What do you think happened to cause this to occur?
SIDE NOTE: SATELLITE COLONIES
Satellite Colony: small bacterial colonies that can surround a large antibiotic-resistant colonyWhy satellite colonies occur: antibiotic resistant bacteria secrete beta-lacatamase to break down ampicillin in the surrounding area allowing non-resistant bacteria to grow in that zone.
What is a protein? A large molecule composed of chains of amino acids
PROTEIN PURIFICATION BACKGROUND
What do proteins do?
PROTEIN PURIFICATION BACKGROUND
HormonesHair and Nails
Structural Support
Enzymes
Muscle Contraction
Antibodies
Receptors, membrane channels
Nutrient Storage
What do proteins do? Inner Life of a Cell -
http://www.youtube.com/watch?v=wJyUtbn0O5Y
Myasthenia Gravis –
PROTEIN PURIFICATION BACKGROUND
How do you make a protein?
PROTEIN PURIFICATION BACKGROUND
DNA mRNA Protein Trait
Why would you ever want to purify a protein? To study the protein To make medicine – insulin, lactase
PROTEIN PURIFICATION BACKGROUND
PROTEIN PURIFICATION BACKGROUND
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use Insulin for diabetics
Things to consider: There are thousands of different proteins in a cell The characteristic of the protein you want to isolate
HOW WOULD YOU PURIFY A PROTEIN
1. Lyse (cut) open the cells.2. Separate the cellular components by size3. Distinguish protein of interest4. Separate protein of interest5. Retrieve protein of interest
See whiteboard
GENERAL STEPS OF PROTEIN PURIFICATION
1. Collect Bacteria Scrape transformed cells into tube with TE Buffer
2. Break Open Cells Add lysozyme to weaken cell wall Snap Freeze to blow up cell
PROTEIN PURIFICATION PROTOCOL STEPS
Lysozyme a naturally occurring enzyme used to weaken the cell wall of e. coli through a hydrolysis reaction–Found in mucus, tear, egg whites
SIDE NOTE: WHAT IS LYSOZYME
Snap Freeze – The process of quickly freezing something
SIDE-NOTE: WHAT IS SNAP FREEZE
3. Centrifuge the cells to separate the heavy cell material from the lighter cell material
PROTEIN PURIFICATION PROTOCOL STEPS
pellet
supernatant
PROTEIN PURIFICATION PROTOCOL STEPS
3. Separate the fluorescent protein from other light cellular debris using column chromatography
3a. Mix supernatant with nickel beads 3b. Pass the
supernatant and nickel bead mixture through the column and into a waste tube
3c. Add elution buffer to column and collect solution in a new tube
SIDE- NOTE: HOW DO THE NICKEL BEADS WORK?
his-his-his-h
is-his-h
is
His- tag: a chain of histidine amino acids
SIDE-NOTE: HOW DO THE NICKEL BEADS WORK?
Ni2+Ni2+
Ni2+
Ni2+Ni2+
Ni2+
Ni2+
The his-tag on the fluorescent protein and nickel bind like 2 magnets
SIDE-NOTE: HOW DOES THE ELUTION BUFFER WORK?
Elution buffer contains a molecule called imidizole that has a stronger attraction to the nickel beads
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
Video: www.youtube.com/watch?v=FUAQKjKT99Y
PROTEINS PURIFIED!
Finished! Now you have a pure sample containing only fluorescent proteins
MAKING THE LAB INQUIRY BASED
What is inquiry? What does Inquiry mean to you?
Student Choice
Students create their own understanding
Topic is relevant and meaningful
Using our understanding of inquiry, how can we incorporate these ideas to make the lab more inquiry based?
20 min brain storm
Share out how to make lab inquiry based
Ask students to come up with their own way of purifying protein
Ask students to figure out why each step is needed. (What is happening at the molecular level?)
Ask students what they would use with this technology. (Ethics discussion)
Do lab twice and change a variable. (we have 7 sets total)
What inquiry is: Student Choice
Students create their own understanding
Topic is relevant and meaningful
___________________
MAKING THE LAB INQUIRY BASED
Agenda for Next Week:
Look at Results of Bacterial Transformation with Students Random Group of Students?
Start Protein Purification Incorporate inquiry based techniques Create a lesson plan Determine your own pacing for your own group Each group will get at least 2 kits
Clean up and Snacks
LESSON PLANNING FOR NEXT WEEK