Download Lisa Byers UNIT 6: Genetic Transformations Unit Plan

NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 1 of 5 Lisa Byers
UNIT 6: Genetic Transformations Unit Plan
Unit Outline:
Lesson 1: Introduction to Transformation and lab set up
Day 1 - Title of Lesson: pGLO Transformation Day 1
Time: One period or 55 minutes.
Prior Knowledge Necessary:
DNA’s role in the continuity of an organism, the process of analyzing DNA through gel electrophoresis, and
basic understanding of heredity (genotypes vs. phenotypes) are all important concepts that should be
covered before this activity.
General Concepts: Bacterial DNA can be transformed through use of a plasmid.
National Science Education Standards
Content Standard A: Science as Inquiry
Abilities Necessary to do Inquiry NSES p.175
“Use technology and mathematics to improve investigations and communications”
“Formulate and revise scientific explanations and models using logic and evidence”
Content Standard C: Life Science
The Cell NSES p.184
“Cells have particular structures that underlie their functions”
“Cell functions are regulated which allows cells to respond to their environment and to
control and coordinate cell growth and division”
The Molecular Basis of Heredity NSES p.185
“In all organisms, the instructions for specifying the characteristics of the organism are
carried in DNA . . . The structural and chemical properties of DNA explain how the genetic
information is encoded in genes and replicated. Each DNA molecule in a cell forms a single
chromosome.”
The Interdependence of Organisms NSES p.186
“Organisms both cooperate and compete in ecosystems. The interrelationships and
interdependencies of these organisms may generate ecosystems that are stable. . .”
AZ High School State Standards
Science Standards
Strand 11: Concept 1: PO 4
Concept 2: PO 5
Strand 2: Concept 1: PO 1
Strand 4: Concept 1: PO 1 & PO 2
Concept 2: PO 1 & PO 2
Concept 3: PO 2
NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 2 of 5 Common Misconceptions:
« Prior beliefs were found to be the major influence in a pupil’s understanding of the experiments that use
sterile control plates in a bacteriological experiment (and) could not be understood if a child’s concepts of
living, size, growth, and reproduction in bacteria are not understood » (Driver, etc al. Pg. 57).
Essential Questions:
- Describe how you could use two LB/agar plates, some E. coli and ampicillin to determine how E. coli
cells are affected by ampicillin.
- What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli
cells?
- What other process have we just learned about that we know can utilize gene therapy?
- What would the gene code for?
- On which of the plates would you expect to find bacteria most like the original non-transformed E. coli
colonies you initially observed? Explain your prediction.
- If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be
located? Explain your predictions.
- Which plates should be compared to determine if any genetic transformation has occurred? Why?
- What is meant by a control plate? What purpose does a control plate serve?
- What else does the plasmid need besides the DNA of the coding enzyme?
Behavioral Objectives:
The students will be able to safely administer all protocol for the laboratory. The students will be able to
correctly answer the essential questions.
The students will be able to describe the role of LB, ampicillin, Arabinose, and pGLO plasmid.
Description of the Lab: This activity is a laboratory activity from BIO-Rad pGLO transformation kit. The
students will be setting up a transformation of DNA to compare with the process and production of
Chymosin.
Incorporation of Inquiry: The activity is a strict procedure that needs to be followed for the intended
outcome to happen. It is on the low end of the inquiry scale, much closer to direct instruction.
Materials:
pGLO Student manual
E. coli starter plate
Poured agar plates
Transformation solution
LB nutrient broth
Inoculation loops
Pipets
Foam microtube holder/float
Container full of crushed ice
Marking pen
Rehydrated pGLO plasmid
42 C water bath and thermometer
37 C incubator
NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 3 of 5 Safety:
Students have a safety contract as agreed to at the beginning of the year. The teacher will review
sterile technique to prevent contamination of lab surfaces and safety. In the contract students
promise to report inappropriate behavior to the instructor immediately, promise not to rough
house in the lab, promise to dress appropriately to lab, promise to not eat or drink anything while
in the lab, promise to know what they are going to do before they conduct a lab.
Textbook:
The diagram and background information comes from Biotechnology Laboratory Manual – Ellyn Daugherty
Biotechnology Explorer pGLO Bacterial Transformation Kit instructors guide – Bio-Rad
Biology, Principals and Explorations – Holt, Rineheart, and Winston
Unifying concepts: Make biotechnology relevant to the students and tie in how it relates to the science they
have already learned. Discover how scientific processes can be used for many different purposes. Visually
seeing how DNA goes to RNA, which then is turned into a protein that is expressed a trait.
Assessments: The students will accurately complete all essential questions including a ticket out the door as
a reflection and assessment.
Real world connections: Bacterial transformation techniques are used in the field of Biotechnology. An
example of bacterial transformations is the process by which Chymosin is created.
Lesson 2: Collection and interpretation of data
Warm up – As students enter they are to answer the following question from their homework from
the previous night:
Which is the best choice for a genetic transformation: a bacterium, earthworm, fish, or
mouse? Describe your reasoning.
5 min - The students will receive their plates to complete Consideration 2 (pg 30 in student manual).
- Describe how you could use two LB/agar plates, E. coli and some ampicillin to determine how
E. coli cells are affected by ampicillin.
- What would you expect your experimental results to indicate about the effect of ampicillin on the
E. coli cells?
- What other process have we just learned about that we know can utilize gene therapy?
5 min - The teacher will randomly call on students for their answers. The teacher will probe for
students to clarify misunderstanding.
5 min – A brief lecture and review of the visual act of transformation (Consideration 3 pg 31).
30 min - The students are to follow the protocol listed below (taken from pGLO Transformation
teacher guide pages 14-15):
NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 4 of 5 1. Label one closed micro test tube +pGLO and another –pGLO. Label both tubes with your group name.
Place them in the foam tube rack.
2. Open the tubes and using a sterile transfer pipette, transfer 250 micro-liters if transformation solution
(CaCl2)
3. Place the tubes on ice.
4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pGLO tube
and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between
your index finger and thumb until the entire colony is dispersed in the transformation solution (with no
floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the
–pGLO tube.
5. Examine the pGLO plasmid DNA solution with the UV lamb. Note your observations. Immerse a new
sterile loop into the plasmid DNA stock tube. Withdraw a loop full. There should be a film of plasmid
solution across the rind. This is similar to seeing a soapy film across a ring for blowing soap bubbles.
Mix the loop full into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on
ice. Also close the –pGLO tube. Do no add plasmid DNA to the –pGLO tube. Why not?
6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so
the bottom of the tubes sticks out and make contact with the ice.
7. While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as follows:
- LB/amp plate: +pGLO
- LB/amp/ara plate: +pGLO
- LB/amp plate: -pGLO
- LB plate: -pGLO
8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the
water bath, set at 42 C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack
so the bottoms of the tubes stick out and make contact with the warm water. When the 50 seconds are
done, place both tubes back on ice. For the best transformation results, the change from the ice (0° C) to
42° C and then back to the ice must be rapid. Incubate on ice for 2 min.
9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a
new sterile pipette, add 250 micro-liters of LB nutrient broth to the tube and reclose it. Repeat with a
new sterile pipette for the other tube. Incubate the tubes for 10 minutes at room temperature.
10. Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100
micro-liters of the transformation and control suspensions onto the appropriate plates.
11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by
quickly skating the flat surface of a new sterile loop back and forth across the plate surface being careful
not to gouge the surface.
12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the
stack and place the stack upside down in the 37° C incubator until the next day.
5 min – Students clean up and may begin to work on their homework.
5 min - Ticket out the door: What would the gene code be for if we did this experiment to help
make cheese production more efficient?
Answer: the enzyme Chymosin
NAU BIOTECH © 2009 Lisa Byers NAU BIOTECH Unit 6 of 12: Genetic Transformations Page 5 of 5 Homework:
- On which of the plates would you expect to find bacteria most like the original non-transformed E. coli
colonies you initially observed? Explain your prediction.
- If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be
located? Explain your predictions.
- Which plates should be compared to determine if any genetic transformation has occurred? Why?
- What is meant by a control plate? What purpose does a control plate serve?
- What else does the plasmid need besides the DNA of the coding enzyme?
NAU BIOTECH © 2009 Lisa Byers