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?
The purpose of the lab was to
change the genotype of e. coli
to change it’s phenotype.
 How did we accomplish this?
REVIEW: HOW WE MAKE
E.COLI GLOW
Plasmid
Uptake of foreign DNA,
often a circular plasmid
Allow bacteria to grow for 1-3
days
DNA  RNA Protein
Bacteria now express cloned fluorescent protein…
REVIEW: 3 AGAR PLATES
What was the purpose of growing bacteria on each plate?
LB/AMP +
LB/No Amp -
LB/AMP 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 antibioticresistant colony
Why satellite colonies occur:
antibiotic resistant bacteria
secrete beta-lacatamase to
break down ampicillin in the
surrounding area allowing nonresistant bacteria to grow in
that zone.
PROTEIN PURIFICATION BACKGROUND
 What is a protein?
 A large molecule composed of chains of amino acids
PROTEIN PURIFICATION BACKGROUND
What do proteins do?
Hormones
Hair and Nails
Muscle Contraction
Structural Support
Enzymes
Antibodies
Receptors, membrane
channels
Nutrient Storage
PROTEIN PURIFICATION BACKGROUND
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?
DNA
mRNA
Protein
Trait
PROTEIN PURIFICATION BACKGROUND
 Why would you ever want to purify a protein?
 To study the protein
 To make medicine – insulin, lactase
PROTEIN PURIFICATION BACKGROUND
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
HOW WOULD YOU PURIFY A PROTEIN
 Things to consider:
 There are thousands of different proteins in a cell
 The characteristic of the protein you want to isolate
GENERAL STEPS OF PROTEIN
PURIFICATION
1.
2.
3.
4.
5.
Lyse (cut) open the cells.
Separate the cellular components by size
Distinguish protein of interest
Separate protein of interest
Retrieve protein of interest
See whiteboard
PROTEIN PURIFICATION PROTOCOL
STEPS
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
SIDE NOTE: WHAT IS LYSOZYME
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 SNAP FREEZE
 Snap Freeze – The process of quickly freezing something
PROTEIN PURIFICATION PROTOCOL
STEPS
3. Centrifuge the cells to separate the heavy cell material from
the lighter cell material
supernatant
pellet
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- tag: a chain of
histidine amino
acids
SIDE-NOTE: HOW DO THE NICKEL
BEADS WORK?
The his-tag on the fluorescent protein
and nickel bind like 2 magnets
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
Ni2+
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.yout
PROTEINS PURIFIED!
Finished! Now you have a pure sample containing only
fluorescent proteins
MAKING THE LAB INQUIRY BASED
Using our understanding of inquiry, how
can we incorporate these ideas to make
the lab more inquiry based?
Student Choice
20 min brain storm
What is inquiry? What does
Inquiry mean to you?
Topic is relevant and
meaningful
Students create their
own understanding
MAKING THE LAB INQUIRY BASED
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
under standing
 Topic is relevant and
meaningful
 ___________________
LESSON PLANNING FOR NEXT WEEK
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