Tuesday: meet @ 6:30-Run DNA lab
Thursday: meet @ 7:00- exam- Chapters 16-21
• 1A2: Natural selection acts on phenotypic variations in populations.
• 1C3: Populations of organisms continue to evolve.
• 3A1: DNA, and in some cases RNA, is the primary source of heritable information.
• 3B1: Gene regulation results in differential gene expression.
• 3C1: Changes in genotype can result in changes in phenotype.
• 3C2: Biological systems have multiple processes that increase genetic variation.
• The student is able to connect evolutionary changes in a population over time to a
change in the environment (1A2 & SP 7.1).
• The student is able to evaluate given data sets that illustrate evolution as an ongoing
process (1C3 & SP 5.3).
• The student can justify the claim that humans can manipulate heritable information
by identifying at least two commonly used technologies (3A1 & SP 6.4).
• The student can predict how a change in a specific DNA or RNA sequence can result
in changes in gene expression (3A1 & SP 6.4).
• The student is able to pose questions about ethical, social, or medical issues
surrounding human genetic disorders [an application of genetic engineering]
(3A3 & SP 3.1).
T152 Investigation 8
• The student can use representations to describe how gene regulation influences cell
products and function (3B1 & SP 1.4).
• The student is able to predict how a change in genotype, when expressed as a
phenotype, provides a variation that can be subject to natural selection
(3C1 & SP 6.4, SP 7.2).
• The student is able to construct an explanation of the multiple processes that increase
variation within a population (3C2 & SP 6.2).
Students will develop the following skills:
• Using sterile technique
• Disposing properly of materials and solutions that come in contact with bacteria
• Transferring bacterial colonies from agar plates to microtubes
• Transforming bacterial cells with plasmid DNA
• Delivering transformed cultures to agar plates
• Applying mathematics to quantify transformation efficiency
Answer the questions for the following lab elements:
Observe the colonies of E. coli grown on the starter LB/agar plate provided by your teacher to glean some information before you determine if any genetic transformation has occurred. What traits do you observe in pre-transformed bacteria? Record your observations in your laboratory notebook.
Some bacteria are naturally resistant to antibiotics, but others are not. How could you use two LB/agar plates, some E. coli, and some ampicillin (an antibiotic) 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? Do you think that exposure to ampicillin will cause the E. coli cells to evolve resistance to ampicillin? Why or why not?
• How will you be able to tell if host E. coli cells have been genetically transformed? (Hint: You will need some information from your teacher about the plasmid you will be using.)
Think about these questions before collecting data and analyzing your results. Be sure to record your answers in your laboratory notebook.
1. On which of the plates would you expect to find bacteria most like the original nontransformed E. coli colonies you initially observed? Why?
2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Again, why?
3. Which plates should be compared to determine if any genetic transformation has occurred? Why?
4. What barriers might hinder the acquisition of plasmids?
5. How can the procedures described above (addition of CI2 and “heat shocking”) help facilitate the introduction of plasmids into the E. coli cells?
Consider the amount of bacterial growth you see on each plate. What color are the colonies? How many bacterial colonies are on each plate? Additional questions you might want to consider include the following:
1. Do your results support your original predictions about the “+ plasmid” transformed E. coli cells versus “- plasmid” nontransformed cells?
2. Which of the traits that you originally observed for E. coli did not seem to become altered? Which traits seem now to be significantly different after performing the transformation procedure?
3. What evidence suggests that the changes were due to the transformation procedures you performed?
4. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?
5. Was your attempt at performing a genetic transformation successful? If so, how successful?
Go over arabinose operon and p-Glo gene expression
By calculating transformation efficiency, you can measure the success of yourtransformation quantitatively.
1. Calculate the total number of transformed cells.
2. Calculate the amount of plasmid DNA in the bacterial cells spread on the LB/amp
plate.a. Calculate the total amount (mass) of plasmid DNA.
DNA in μg = (concentration of DNA of μg/μL) x (volume of DNA in μL)
b. Calculate the fraction of plasmid DNA that actually got spread onto
the LB/amp plate.
Fraction of DNA used = Volume spread on the LB/amp plate (μL)
Total sample volume in test tube (μL)
Calculate the micrograms of plasmid DNA that you spread on
the LB/amp plate.
DNA spread in μg = Total amount of DNA used in μg x fraction of DNA used
What does this number tell you?
3. Calculate transformation efficiency. Page 108
Look at your calculations. Fill in the blanks with the correct numbers.
Number of colonies on the LB/amp plate:
Micrograms of plasmid DNA spread on the plate:
Now calculate the efficiency of the transformation.
Transformation efficiency = Total number of colonies growing on the agar plate
Amount of DNA spread on the LB/amp plate (in μg)
4. What does this mean?
a. Report your calculated transformation efficiency in scientific notation.
b. What does your calculation of transformation efficiency mean?
c. Biotechnologists generally agree that the transformation protocol that you have
just completed has a transformation efficiency of between 8.0 x 102 and 7.0 x 103
transformants per microgram of DNA. How does your transformation efficiency
compare? What factors could explain a transformation efficiency that was greater
or less than predicted?
■■Evaluating Results page 109
1. What are some challenges you had in performing your investigation? Did you make
any incorrect assumptions?
2. What are some possible sources of error in the transformation procedure? If you had
to repeat the procedure, what are ways to minimize potential sources of error?
3. Were you able to perform without difficulty the mathematical routines required to
calculate transformation efficiency? Which calculations, if any, were challenging or
required help from your classmates or teacher?
4. Can you suggest other preliminary activities that would have better prepared you to
tackle the investigation?
5. Does a bacterial cell take in a plasmid with genes the cell already possesses? If so,
would this affect your calculations?
■■Designing and Conducting Your Investigation page 109
Think about these questions again for a minute.
• What causes mutations in bacteria? Can mutations affect plasmids? How would you
be able to tell if any observed changes in phenotypes are due to the expression of
genes carried on plasmids and are not attributed to a possible mutagen?
• Do bacteria take up more in plasmid in some conditions and less in others? What
conditions favor uptake, and which ones inhibit it?
• What other questions do you have about plasmids and transformation?
■■Where Can You Go from Here? page 110
The background to this investigation asks you to think about several applications
of genetic transformation, including genetically modified food and possible ethical,
social, or medical issues raised by the manipulation of DNA by biotechnology. Why are
these “issues”? What questions are posed by genetic engineering? In terms of what you
have learned about biotechnology, how would you respond to the quote from Michael
Crichton’s novel and film Jurassic Park: “Just because science can do something doesn’t
mean that it should”?
BIOTECHNOLOGY: RESTRICTION ENZYME ANALYSIS OF DNA*
How can we use genetic information to identify and profile individuals?
Answer questions posed in activities I-III.-