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 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

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 fl uorescent 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 fi gure 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