EDS 198 ADAPTING LABS FOR INQUIRY

 Lab Review and Analysis of Results  Adapting Lab for Inquiry  Protein 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 colony Why 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 Hormones Hair and Nails Structural Support Enzymes Muscle Contraction Antibodies Receptors, membrane channels Nutrient Storage

What do proteins do?  Inner Life of a Cell  Myasthenia Gravis – PROTEIN PURIFICATION BACKGROUND

How do you make a protein? PROTEIN PURIFICATION BACKGROUND DNAmRNAProteinTrait

 Why would you ever want to purify a protein?  To study the protein  To make medicine – insulin, lactase 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 size 3.Distinguish protein of interest 4.Separate protein of interest 5.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-his-his-his His- tag: a chain of histidine amino acids

SIDE-NOTE: HOW DO THE NICKEL BEADS WORK? Ni 2+ 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 Ni 2+ Video: ube.com/ watch?v= FUAQKjKT 99Y

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