MODULE 2
Objective 2.1 Lesson E
GFP Transformation
Genetic Manipulations
Course
Advanced
Biotechnology
Unit
DNA
Technology
Essential
Question
How is foreign
DNA genes
taken up by
organisms and
expressed?
TEKS
130.364 1A-K,
2E-J, 7A, 7B,
7F, 7G, 9A, 9C,
9F, 10A,
10B,11A, 11B
Rationale
Is it possible to manipulate bacteria to become protein production factories?
Can diabetics control blood glucose with insulin produced by bacteria? How
cool would it be to take advantage of this microorganism’s sophisticated
makeup, short doubling times and cheap growth media to mass-produce
medically and commercially useful proteins? All of these are possible with a
few simple genetic manipulations.
Objectives
Students will:
 Practice formulating hypotheses, predictions, and experimental design.
 Describe the principles of bacterial transformation.
 Explain the procedure for gene transfer using plasmid vectors.
 Induce the transfer of the GFP gene (in a plasmid) into E. coli.
 Describe the traits carried by the GFP gene.
 Describe how to activate (“turn on”) the GFP gene.
 Describe how to recognize the transformed cells (from this lab).
 Perform a bacterial transformation.
 Determine the degree of success in your efforts to genetically alter an
organism.
TAKS
Science 1A, 2A,
2B, 2C, 2D, 6A
Prior Student
Learning
Recombinant
DNA, bacterial
structure,
protein
expression
Estimated Time
4 days
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Key Points
I. Dolan Center Information and Expected Results:
http://labcenter.dnalc.org/labs/transformation/transformation_h.html
II. Videos on Lab Procedure and Expected Results:
o Part 1: http://www.youtube.com/watch?v=Jyfi5e2JoYc
o Part 2: http://www.youtube.com/watch?v=7zJXe9rZh8&feature=related
Engage
1. Assign each lab group one of the recombinant product below:
 Human Insulin
 Recombinant Growth Hormone
 Spider Silk
 Golden Rice
 Edible Vaccine
 Bt Crops
2. Give each lab group 20 minutes to use the Internet to
research the science behind each product, its uses and
common characteristics.
Example:
Golden Rice
Science behind it
Genes for making beta carotene are taken from
daffodils and inserted into the genome of a strain of
rice.
Characteristics
Contains beta-carotene which forms Vitamin A
Uses
To protect undernourished people caused by a lack of
Vitamin A
3. Allow groups to present to the class.
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Activity
There are several green fluorescent protein transformation kits
available through your district approved science vendor.
1. Pre-Lab Set Up: Have students set up the lab stations, materials
and equipment the day before for a lab grade. An Instructor’s Advance
Preparation will come with any GFP Bacterial Transformation Kit.
Below are sample lab group assignments to perform:
a. Set up Student Workstation
b .Set up Instructor’s Workstation
c. Prepare Agar Plates
d. Label Agar Plates
e. Aliquot Transformation solution
f. Prepare Starter Plates
2. Students complete “Virtual Bacteria Transformation” worksheet. This
activity will give student’s background information about how scientist
create and identify recombinant plasmids. The lab procedure is similar to
the wet lab they will perform in the classroom. Answer key provided.
3. Student will need some specific background information and the green
fluorescent protein and how it is selected for in the lab before beginning
this lab. Kits should come with student background reading, teacher
instructions. Some even provide power points for direct teach. Dolan
Science also has a background reading section at
http://labcenter.dnalc.org/labs/transformation/transformation_d.html.
There are two approaches that can be used for this lab:
I.
Experimental Design Approach resulting in a Formal Lab Report.
a. Have students use Lab Write to guide them through the
writing process. This software has many different features to
help students write effective lab reports. There is also a
teacher section which provides materials that can be used to
introduce students to Lab Write and how to use it.
b. Direct students to DNALC Lab Center: Bacterial
Transformation. At this site students have access to
background readings on GFP, student protocols, pre-lab
worksheets, post lab worksheets, applications and further
exploration exercises. Allow students to explore this site on
their own to find the information and materials needed to
complete a lab report using Lab Write.
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c. Steps to implement:
 Assign student the pre-lab section in Lab Write.
 Students complete the lab protocol provided by the
transformation kit.
 Assign students the lab section in Lab Write.
 Students record results using post-lab section in Lab
Write.
 Students peer grade using Lab Write grading rubric.
II. Descriptive Approach resulting in completion of a handout.
 Students complete all parts of the “Bacterial Transformation”
Handout. This handout pre-lab, lab, analyzing the results,
applications and an extension section.
Materials
 Handout: “Virtual Bacterial Transformation”
 Handout: “Virtual Bacterial Transformation” Answer Key
 Handout: “LAB: bacterial Transformation”
 Handout: “Formal Lab Report Guidelines”
 Science Kit: GFP Transformation from district approved
science supply vendor.
Accommodations for Learning Difference
 Visit the Special Populations section of the CTE Career and
Technical Education Website: http://cte.unt.edu/special-pops.
Texas College and Career Readiness Standards
I. Nature of Science: Scientific Ways of Learning and Thinking
A4, B1, C1, C2, C3, D3, E1, E2
II. Foundation Skills: Scientific Applications of Mathematics
A1, A2, A5, A6, A7, B1, B2, F1, F2, F3
III. Foundation Skills: Scientific Applications of Communication
B1, B2, B3, C1
VI. Biology
A1, D3
Copyright © Texas Education Agency 2012. All rights reserved.
Virtual Bacterial Transformation
Name:____________________
Go to site below to help you complete this document.
http://www.montereyinstitute.org/courses/AP%20Biology%20I/course%20files/multimed
ia/chapter9virtuallab02/lessonp.html
Overview
1. Define biotechnology2. List the four steps involved with molecular cloning.
3. What are the three techniques used commonly in biotech labs described by the
video?
4. Restriction digestion is a laboratory technique in which proteins called
____________ ____________ are used to cut DNA at specific sequences of
nucleotides.
5. _____ ________________ is a laboratory technique in which macromolecules
or fragments of macromolecules are separated according to their migration in an
electric field.
6. What sized molecules move faster in the electric field?
7. Define bacterial transformation-
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8. Create a recombinant plasmid by completing the following steps:
A. Draw a plasmid.
B. Draw a general diagram of a plasmid after a small DNA fragment has been cut
from the plasmid by a restriction enzyme. In the diagram, be sure to include the
DNA fragment that will eventually get replaced by foreign DNA. Label the
plasmid, the DNA fragment, and all sticky ends.
Copyright © Texas Education Agency 2012. All rights reserved.
C. In the left section below, draw a snapshot figure just before DNA ligase joins
the foreign DNA segment to the plasmid. Label DNA ligase, foreign DNA,
plasmid, and ALL sticky ends. In the right section below, draw the recombinant
(completed) plasmid after DNA ligase has finished its job. For each section,
highlight the foreign DNA and the plasmid with different colors.
I.
II.
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Plasmid Analysis
Fill in the data table with the correct values from the Gel Electrophoresis step.
Gel Electrophoresis Data
Lane
Sample
1
Ladder
2
1
3
2
4
3
Fragments (kB)
9. Using the table below and the gel electrophoresis data, identify which plasmids
are present in each sample.
Sample 1=___________
Sample 2 =__________
Sample 3 =__________
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Bacterial Transformation
10. Calcium ions are used to make ___________ cells. This treatment enhances the
uptake of DNA for these cells.
11. What additional procedure is necessary to allow bacteria to take up plasmid
DNA?
12. Only some competent bacterial cells have successful uptake of the plasmid
DNA. How is ampicillin used to identify these desired bacterial cells?
13. An indicator called X-GAL is used to identify which bacterial cells do and do not
contain the insulin gene within their plasmid. Specify which color identifies the
bacterial colony containing the insulin gene and the color for the colony without
the insulin gene?
14. Define transformation efficiency-
Copyright © Texas Education Agency 2012. All rights reserved.
Virtual Bacterial Transformation Answer KEY
Overview
Define biotechnologyThe use of living things or parts of living things to create products useful to society.
List the four steps involved with molecular cloning.
1) Isolate the insulin gene.
2) Insert insulin gene into another piece of DNA.
3) Insert recombinant DNA into bacterial cell.
4) Grow bacteria and allow them to produce insulin.
What are the three techniques used commonly in biotech labs described by the video?
Restriction digestion, gel electrophoresis, and bacterial transformation
Restriction digestion is a laboratory technique in which proteins called restriction enzymes are used to
cut DNA at specific sequences of nucleotides.
Gel electrophoresis is a laboratory technique in which macromolecules or fragments of
macromolecules are separated according to their migration in an electric field.
What sized molecules move faster in the electric field?
Smaller DNA fragments and molecules
Define bacterial transformation-the process by which bacteria are changed by the uptake of foreign
DNA.
Create a recombinant plasmid by completing the following steps:
Copyright © Texas Education Agency 2012. All rights reserved.
1) Draw a plasmid.
OR
2) Draw a general diagram of a plasmid after a small DNA fragment has been cut from the
plasmid by a restriction enzyme. In the diagram, be sure to include the DNA fragment that
will eventually get replaced by foreign DNA. Label the plasmid, the DNA fragment, and all
sticky ends.
Sticky ends
DNA fragment
Sticky ends
Plasmid
OR Same but using simple
inner/outer circle plasmid
Copyright © Texas Education Agency 2012. All rights reserved.
3) In the left section below, draw a snapshot figure just before DNA ligase joins the foreign
DNA segment to the plasmid. Label DNA ligase, foreign DNA, plasmid, and ALL sticky
ends. In the right section below, draw the recombinant (completed) plasmid after DNA
ligase has finished its job. For each section, highlight the foreign DNA and the plasmid
with different colors.
Foreign DNA
Sticky
ends
I.
Sticky ends
DNA
Ligase
II.
DNA
Ligase
Plasmid
Plasmid Analysis
Fill in the data table with the correct values from the Gel Electrophoresis step.
Gel Electrophoresis Data
Lane
Sample
Fragments (kB)
1
Ladder
2
1
3
2
2.5
1.1
4
3
1.6
1.1
2.8
Using the table below and the gel electrophoresis data, identify which plasmids are present in each
sample.
Copyright © Texas Education Agency 2012. All rights reserved.
Sample 1=
Sample 2 =
Sample 3 =
B
C
A
Bacterial Transformation
Calcium ions are used to make competent cells. This treatment enhances the uptake of DNA for these
cells.
What additional procedure is necessary to allow bacteria to take up plasmid DNA?
Heat shock
Only some competent bacterial cells have successful uptake of the plasmid DNA. How is ampicillin
used to identify these desired bacterial cells?
Only the plasmid DNA contains the ampicillin resistance gene amp. The bacteria that do not contain
the plasmids will die from ampicillin because they do not have the ampicillin resistance gene. When
ampicillin is applied, only the bacterial cells that have successfully accepted the plasmid DNA will
survive.
An indicator called X-gal is used to identify which bacterial cells do and do not contain the insulin gene
within their plasmid. Specify which color identifies the bacterial colony containing the insulin gene and
the color for the colony without the insulin gene?
White-Colony with insulin gene
Blue-Colony without insulin gene
Define transformation efficiency-the ratio of the number of colonies formed to the amount of plasmid
DNA used
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LAB: Bacteria Transformation
I.
Pre-Lab
Name :_______________________
Answer the questions and fill in the table in the space provided below.
Complete the Hypothesis, Predictions, and Experimental Design sections during
the first lab period. The Results section will be completed after we analyze the
data next week.
Use the DNALC Lab Center: Bacterial Transformation Website to help you
answer these questions.
Hypothesis
Formulate a hypothesis on which this investigation is based, of how E. coli
cells can be transformed by the pGLO plasmid.
Predictions
Prepare and complete the table below to indicate what you predict will
happen on each of the four agar plates. (Will E. coli grow on these plates?
Will the E. coli have any special properties compared to wild type?)
Plate
Plasmid?
LB/amp
+DNA
LB/amp/ara
+DNA
LB/amp
-DNA
LB
-DNA
Growth (G)
No Growth (NG)
Other properties?
Note: LB is the nutrient mixture that is added to the plate agar to feed the bacteria.
Experimental Design
Explain the experimental design:
1. What is/are the independent variable(s) in this experiment?
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2. What is/are the dependent variable(s)?
3. Which plates will serve as control plates? Do you expect cells to grow on
these plates? Why or why not? What is the purpose of these controls?
4. Define plasmid.
II.
LAB: Your instructor will provide you will the lab protocol. Create a flowchart
below of the steps you will take in this lab.
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III. Analyzing the Results
Results
1. In the table below, fill in your observations after examining your plates
under both normal and UV light.
Number of
Plate
Plasmid?
Other Properties?
Colonies?
LB/amp
+DNA
LB/amp/ara
+DNA
LB/amp
-DNA
LB
-DNA
2. Was your genetic transformation successful? How do you know?
3. Are your results consistent with the predictions you made in the table
on the previous page? If not, why?
4. Consider the following two pairs of plates. What do the results obtained from
these plates tell you about your experiment?
a. -DNA LB and -DNA LB/amp
b. +DNA LB/amp and -DNA LB/amp
5. After examining your results, would you revise your hypothesis? If so, restate
your hypothesis below.
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IV. Applications
Discuss the various uses of GFP as a reporter protein. Students can look
up scientific articles about scientists that use GFP in their experiments.
Research other bioluminescent organisms, and describe how they have
been incorporated into biological research.
V.
Extension:
How could you confirm which bacterial cells contained the GFP gene?
Using your results plates, design an experiment that could help you
answer this question? Briefly describe what you will do. Do it. Record
your results below.
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Transformation Lab Report Requirements
I. Title of Experiment
 Determine the lab’s independent & dependent variables, then generate an appropriate title!
(+2)
II. Introduction
• Introductory paragraph describing key terms associated with experiment: plasmid,
transformation, competent (+3)
III. Hypothesis
total)
• Formulate and make predictions(+2)
• Discuss your experimental design (+3)
(5 pts
IV. Methods/Procedure
total)
(15 pts
• Briefly describe/summarize experimental procedure and include the following information:
(+5)
o Explain importance/role of –pGLO tubes in experimental design
o Explain importance/role of CaCl2 in experimental method
o Explain importance of and parameters for successful heat shock
• For each plate, identify purpose/role in experimental design and provide an hypothesis for
expected results: -pGLO: LB plate, -pGLO: LB/amp, +pGLO: LB/amp, +pGLO:
LB/amp/ara (+8)
• Explain how transformation efficiency is calculated (+2)
V. Results All data tables must have a title AND all columns must be labeled at the top (20 pts
total)
• Table 1: Observations (quantitative and qualitative) for E. coli starter plate (+5)
• Table 2: Transformation experiment results (+10) Do NOT use the pre-lab table you were
given!
• Transformation efficiency calculation for your group’s results for +pGLO/amp plate: (+5)
o Present calculations in logical, step-by-step manner.
o Explain what you are calculating in each step.
o Include units for all numbers within calculations & units for final answer!
VI. Conclusion
total)
• Was your transformation experiment successful? How do you know? (+5)
• Are your results consistent with the predicted results? If not, why? (+2)
• Would you revise your hypothesis? (+3)
(10 pts