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BIO-RAD
pGLO Bacterial Transformation Kit
for General Biology
Catalog #17006991EDU
Student Guide
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Student Guide
Results Analysis
Gather your plates from the previous activity. Keep the lids on while you inspect them. Use a UV light
to check for fluorescence.
B. In Table 2 below, sketch and describe your results from the transformation activity. Draw
any growth and include labels.
Table 2. Results from the bacterial transformation activity.
LB plate (LB)
+pGLO
LB plate with ampicillin (LB/amp)
–pGLO
+pGLO
Description
Description
Differences from prediction in Table 1
Differences from prediction in Table 1
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–pGLO
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Student Guide
C. In Table 2, describe any differences between your predictions in step A and your actual
results.
D. Before you started the transformation activity, what did both plates have in common?
E. Before you started the transformation activity, what was different between the two plates?
Look at the bacterial growth on each plate. You may see two types of growth: individual circles of
bacteria called colonies and areas where there are so many colonies that they merge together. That is
called a bacterial lawn.
F. On your plates, where was there no bacterial growth?
G. On your plates, where did bacteria grow as colonies?
H. On your plates, where did bacteria grow as a lawn?
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Student Guide
I.
Write a claim about how the presence or absence of ampicillin impacted bacterial growth.
Use your observations from both plates as evidence to support your claim. Use complete
sentences.
J. Do you think the gene for green fluorescent protein was successfully transformed into any
of the bacteria on either of your plates? Justify your answer using your observations,
the pGLO plasmid map in Activity 1, Part 3, and any other resources available. Use
complete sentences.
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Student Guide
Part 2. Switch ON the GFP Gene
Background
In all organisms, the expression of genes is tightly controlled. Some genes might be turned ON, or
expressed, while other genes might be turned OFF. When a gene is turned ON, RNA polymerase
binds to the promoter sequence ahead of the gene and then continues to transcribe the gene into
messenger RNA (mRNA).
RNA polymerase
Direction of transcription
Promoter
Gene sequence
Some genes are always ON. For example, on the pGLO plasmid, Ampr is designed to always be ON.
Other genes are turned OFF and require an input to switch ON. The pGLO plasmid also includes a
gene called araC (Figure 6) that codes for the protein of the similar name, AraC. AraC acts as an ON/
OFF switch. When arabinose is present, AraC switches the GFP gene ON. Without arabinose, AraC
switches the GFP gene OFF.
araC
Codes for AraC, which
acts as a ON/OFF switch
for GFP.
pGLO
GFP
Codes for green
fluorescent protein
Ampr
Provides resistance to
ampicillin, an antibiotic
Fig. 6. Simplified map of the pGLO plasmid showing the araC gene.
17
Student Guide
K. Refer back to your results from Activity 2. Talk with your group about whether the GFP gene
was switched ON or OFF in E. coli on your plates and how you know. Describe your thinking
in complete sentences.
Experimental Design
With your group, design an experiment using the available materials to determine whether the bacteria on
your plates from Part 1 have the GFP gene.
Available Materials
Quantity
Common workstation
Quantity
Results plates from Part 1
1
Arabinose solution and shared transfer pipet (per group )
250 µl
UV light
1
Incubator oven set to 37°C (recommended)
1
(LB plate and LB/amp plate)
Marking pen
1
L. Describe the steps of your experiment below. A drawing may be helpful. Remember to
include labels.
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Student Guide
M. Sketch and describe your expected results in Table 3.
Table 3. Predicted results for the GFP switch activity.
LB plate (LB)
+pGLO
Description
LB plate with ampicillin (LB/amp)
–pGLO
+pGLO
–pGLO
Description
N. Explain how your expected results will allow you to determine whether the bacteria on
your plates from Part 1 have the GFP gene.
O. Once you have reviewed your plan with your instructor, carry out your experiment.
19
Student Guide
Results Analysis
P. Examine your plates in regular light and then in UV light. Sketch and describe your
observations below.
Table 4. Results from the GFP switch activity.
LB Pplate (LB)
+pGLO
LB plate with ampicillin (LB/amp)
–pGLO
+pGLO
Description
Description
Differences from prediction in Table 3
Differences from prediction in Table 3
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–pGLO
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Student Guide
Q. Why did some bacteria glow green while others did not?
R. Write a claim about how arabinose influenced the expression of the GFP gene. Use your
observations from both plates as evidence to support your claim. Use complete sentences.
S. Look back to your model in Activity 1, Part 1. Revise the model to include all of the new
information you have gathered. The model should now include:
• the bacterial cells, pGLO plasmid, GFP, arabinose, and ampicillin
• arrows to indicate actions that occur
• labels for all the components
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Student Guide
Activity 3
Bacterial Transformation Design Challenge
Background
In the pGLO plasmid system, you can think of GFP as an output in response to arabinose as an input.
When you add arabinose (input) to bacteria that have been transformed with the pGLO plasmid, they
begin to produce GFP (output). By creating a plasmid with different genes than those on pGLO, E. coli
can be engineered to be responsive to different inputs and produce different outputs. Bacteria that are
engineered to be responsive to an input are called biosensors.
Table 5. Example inputs and outputs.
Inputs
Outputs
Light responsive
Cold responsive
Drought responsive
Sucrose responsive
Low oxygen responsive
Heavy metal responsive (like lead or arsenic)
Changes color
Produces a smell (bananas, cherries, mint)
Makes a protein (insulin, cancer drug)
Captures toxins (lead, arsenic, mercury)
Table 5 provides only a small list of example inputs and outputs that engineers might use when
designing a biosensor. There are many others that you can investigate on your own. Now it is your turn
to develop an idea for a biosensor that could solve a real world problem.
Challenge Instructions
Design and propose a bacterial system that could be used as a biosensor to solve a real-world
problem. Use the design proposal template to structure your idea.
In your proposal you must:
• Identify a problem that could be solved using a biosensor. Remember that biosensors are great
for detecting something. What would be useful to detect?
•
Explain why it is important to solve this problem
•
Explain the solution by:
• drawing a model of the system, including the necessary elements of the plasmid
• describing how the system would be implemented
• describing the inputs and outputs of the system
•
Identify two strengths and two limitations of this solution. Consider some of the following:
• public opinion — what would the public think about this?
• scaling up — what would have to be done to make enough of the product?
• concerns — what are some possible benefits of the solution? What are some of the
potential risks?
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Student Guide
Design Proposal Template
Define the Problem
What is the problem?
Who does the problem affect?
Why is this problem worth solving?
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Student Guide
Design the Solution
Describe your solution.
Explain how your solution will solve the problem.
Identify two strengths and two limitations of this solution.
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Student Guide
Label the sequence components you would add to your
plasmid design. Use generic language.
List the plasmid components here and explain
how each of them works in your solution.
Control element
(input control)
Plasmid DNA
Resistance
Response gene
(output)
Draw a diagram that describes how your system will be implemented to solve the problem. Include labels.
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Student Guide
Bacterial Transformation Design Challenge Rubric
Question
Novice
Developing
Proficient
What is the problem?
Details of the
problem are missing;
demonstrates little
understanding of the
problem.
Explains some details of the problem
but includes irrelevant details
or is missing important details;
demonstrates some understanding
of the problem.
Clearly explains relevant details of
the problem; demonstrates a strong
understanding of the problem.
Who does the problem
affect?
Does not describe the
affected individuals with
any relevant details.
Describes the affected individuals
in some detail but is missing key
details or includes irrelevant details.
Clearly describes the individuals
affected by the problem using
information that is relevant to the
problem.
Why is this problem
worth solving?
Does not explain the
potential impact of
solving the problem.
Explains the potential impact of
solving the problem in some details
but does not provide evidence or
justification.
Clearly explains the potential impact
of solving the problem and provides
quantitative and/or qualitative
evidence to justify the importance.
Describe your solution.
Does not explain all
the components of the
system and is missing
important details.
Explains the system but is missing
some components or important
details.
Clearly describes the inputs,
outputs, and context of the system;
describes how the components
work together.
Explain how your
solution will solve the
problem.
The solution does not
connect to the problem
or the connection is not
explained.
Explains some connections between
the problem and the solution but
does not explain how the solution
will be successful for the intended
audience.
Clearly explains the connection
between the solution and the
problem; provides details about how
the solution will be successful for
the intended audience.
Identify two strengths
and two weaknesses
of your solution.
Does not identify
real strengths and
weaknesses
Identifies strengths and weaknesses
but some are trivial or not well
explained.
Identifies and explains two real
strengths and two real weaknesses
of the proposed solution.
Solution diagram
Some plasmid
components are
missing, and the
diagram is not
complete.
Includes all the necessary plasmid
components but some are not well
described or are not included in the
diagram
All the necessary plasmid
components are included,
described in detail, and included in
a detailed diagram.
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Student Guide
Glossary
Aequorea victoria — a jellyfish species whose chromosome naturally has the gene for green fluorescent
protein and glows green under UV light.
Agar — a gelatinous substance derived from seaweed that can be used to make solid media for
bacterial growth.
Ampicillin — an antibiotic in the penicillin family that is commonly used in laboratories. Bacteria with the
Ampr gene are resistant to ampicillin and continue to grow in its presence.
Ampr — a gene that provides resistance to ampicillin.
Antibiotic resistance — a trait that allows bacteria to grow in the presence of an antibiotic that normally
prevents bacterial growth.
Arabinose — a carbohydrate found in plants that can be a food source for bacteria. It can switch on
gene expression that is controlled by araC.
AraC — an activator protein that acts as an ON/OFF switch for the PBAD promoter.
araC — a gene that codes for the protein AraC.
Bacterial colony — a circular mass of genetically identical bacterial cells growing on solid media.
Bacterial lawn — a growth pattern in which individual bacterial colonies have merged together.
Bacterial transformation — a process for inserting plasmid DNA into bacteria.
Biosensor — a device that use biological organisms or materials to detect a certain input.
Escherichia coli (E. coli) — a rrod-shaped bacterium commonly used in laboratories and genetic
engineering.
Fluorescence — the appearance of glowing or giving off visible light often caused by exposure to
UV light.
Gene expression — the general process in which the instructions in a gene are used to make a protein
or other molecule.
Green fluorescent protein (GFP) — a protein that fluoresces green when exposed to UV light.
LB (Lysogeny Broth, also known as Luria-Bertani broth) — a mixture of nutrients that is commonly
used to grow bacteria.
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Student Guide
mRNA (messenger RNA) — a type of RNA that is transcribed from DNA and is involved in protein
synthesis.
PBAD — a promoter region that can be regulated by AraC.
pGLO — a specific plasmid that includes the GFP gene.
Plasmid — a circular piece of DNA that carries genes and can be inserted into a bacterium in a process
called bacterial transformation.
Plasmid map — a diagram that includes annotations about the genes and genetic features of a plasmid.
Promoter — a DNA sequence where RNA polymerase attaches to begin transcribing a gene.
RNA polymerase — a protein that transcribes DNA sequences into mRNA.
Transcription — the process in which RNA polymerase synthesizes mRNA using a DNA sequence
as a template.
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1120 Sig 0220
Bio 256 General Genetics Formal Lab Report
Title
The title of a research manuscript should be a short declarative statement about the question
answered by the research.
Introduction
Please note this is a formal scientific, written assignment, so both the grammar and scientific
information must be correct. Scientific reports are factual and written in the passive tense. I’m
sorry to say that humor must be left by the wayside.
The introduction starts broadly and focuses on the specific necessity of your hypothesis. Try to
be as concise and specific as you can while still catching the reader’s interest. Begin by
introducing the system in general and what’s known about it (this will require some outside
research on your part.). Give definitions for all the terms you introduce (consider your
audience) and use examples to clarify points.
You should then introduce the problem you are addressing (genetic modifications and gene
regulation). You should explain why this is of interest in such a way that your hypothesis is a
natural consequence of the introduction. The reader should think to themselves, “Of course the
question should be answered! It follows directly from what the author has told me”! Your
hypothesis and its predictions should then be specified.
Key Points:
• Catch the readers interest!
• Make it an introduction
• Use examples
• Give definitions
• Talk about why this is of interest (i.e., why you are doing this experiment).
• Introduce concepts used in the paper
• This section must include either a statement of your research question or a statement of
your hypothesis and prediction.
Methods
The methods should provide sufficient detail so that a reader could repeat your study. Science,
after all, depends on repeatability! The methods section should NOT be a simple laundry list of
your materials, coupled with a short listing of your procedures. That strategy typically results in
confusion, and makes your paper longer than it needs to be. Start with a description of your
experimental system, such as the genes you will study or the organism on which you will
perform tests. Next, write a paragraph (or 75 paragraphs) describing what you did and how you
organized your experiments (i.e., your experimental design). A full description of your
experiments will naturally include the materials you used. Be sure to include how you plan to
analyze your data. Be as complete and as concise as you can and write using the past tense (you
already did the experiment, you are not telling your reader what to do, but what you actually
did!).
Key Points:
• This is NOT a list.
• This is a paragraph or paragraphs completely describing your experimental design and how
you created and collected your data.
• It should be in the past tense or passive tense.
• It is not sufficient to say “See the lab manual.”
Results
In this section you should report your data without drawing conclusions (just the facts, ma’am).
A good way to do this is to present your most important results first, in paragraph form, placing
your well- constructed graphs and tables within this section as supplemental material. These
act essentially as arguments for the reader to see for themselves what the data looks like, while
you, in the text, explain what the reader is seeing in the graph or table. This means that the
words you use to describe your results should only use the words of the independent and
dependent variables on your photos and tables. Be aware that tables and images cannot be
presented without accompanying text to explain them, though they should be complete
enough to make sense without your explanation. Be sure to include a summary caption
highlighting differences between images. Similarly, a table should include the names and units
of variables, a title and explanatory caption, and any other appropriate explanatory
information.
Key points:
• Describe your results
• Place photos and tables within the results section
• Use your text to describe your photos & tables
• Make sure images are complete
• Raw data, or data not summarized as means of several measurements, does not belong in a
results section
Discussion
Use this section to interpret your results or draw logical conclusions from your results, without
repeating your results (this wastes space). Explain how your results fit with the research you
mentioned in your introduction, or other studies you’ve come across. Go back to your
introduction (if it was a good introduction) and address the questions and hypotheses that you
introduced. Discuss what you can conclude from your studies as well as any caveats that you
need to address. Were there any methodological problems? Do NOT say that something must
have gone wrong if your results are different than your expectations. What do your results
mean in light of the context addressed by these experimental results? Based on your findings
you should be able to provide suggestions for future research: this is where your sensor
proposal fits. Based on your findings from the transformation, how could this process be used
in a real-world setting?
Key Points:
• Interpret your results
• Address your hypothesis
• Discuss caveats & future research
• Do not be surprised if your results don’t match your expectations
References
You need a minimum of three references, and one of them must be a primary source article.
Your textbook and lab book are valid sources, but neither is a primary research paper.
Appendix
–Raw data tables go here, as needed.
——————————————————————————————————————–GRADING RUBRIC FOR FORMAL LAB REPORTS
Excellent (15 pts)
Title
Introduction
Materials
and Methods
Results
Discussion
References
Provides background
and rationale for the
experiment and
variables, including a
primary research
reference
Includes reactants,
methodology, controls
in paragraph form
Properly labeled photos,
with written summaries
of the genetic
components that were
recombined
Clear interpretation of
data, including rejection
or support of hypothesis
and prediction; concise
summary, direction for
future research
One primary research
reference and two
secondary sources cited
in APA format
Above Average
(10 pts)
Average (5 pts)
Developing (0
pt)
Specific, descriptive
title
Unclear title
Missing title
Provides basic
background with
limited rationale and
includes a primary
research reference
Provides basic
background with
limited or no
rationale and lacks
a primary research
reference
Includes reactants
and methodology
Provides basic
background and
limited or no
rationale and lacks
a primary research
reference
Includes either
reactants or
methodology
Photos or written
summary
Includes reactants or
methodology and
controls
Photos, with written
summaries of the
genetic components
that were
recombined
Clear interpretation
of data, including
rejection or support
of hypothesis and
prediction;
directions for future
research
Three secondary
sources cited in APA
format
A written summary
of the genetic
components that
were recombined
Interpretation of
data with limited
analysis
Brief summary of
conclusion
Two secondary
sources cited in
APA format
One source cited in
APA format

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