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pGLOTM Transformation Experiment Student Report Sheet
Name: __________________________________________
Prelab: ____ / 5
Data: _____ / 4
Analysis section:
Period: _____
/16
Total Score: ______/ 25
Prelab:
1) What is the objective of this experiment?
2) How will you know if your E. coli have been transformed?
3) What role does the CaCl2 solution play in the transformation process?
4A) Why are the CaCl2 solution, plasmid and cells chilled in an ice bath initially?
4B) Why is the solution of CaCl2 and cells heat shocked for 50 seconds? What happens
during this time?
Data Collection:
- Sketch the contents of each plate below and describe the color and number of colonies
present.
Analysis of results:
1A) Which are the experimental control plates in this experiment? What key
component is absent from the control plates?
1B) What is the purpose of the control plates?
2) Are there any obvious differences in color, size, or growth patterns of the control
plates compared to the transformed plates?
3A) Why would you expect that to be no growth on the control plate –pGLO/
LB/amp?
3B) If fact growth were observed on this plate, what is the most likely explanation
(assume that all lab protocols were carried out correctly and no contamination from
outside bacteria occurred) for the growth?
4A) Which colonies glow under black light?
4B) What substance is fluorescing when exposed to black light: the E. coli Cell
membranes, the pGLO DNA or the pGLO gene product (protein)? Clearly explain how
your direct experimental observations support your conclusion.
5A) What substance is apparently necessary (must be present in the environment) to turn
on the expression of the pGLO gene?
5B) What is the advantage to cells to be able to regulate, i.e. turn on or off, the expression
of specific genes? (In other words, why are genes always turned on or expressed at the
same level?)
6) Based on your observations, which of the following scenarios are consistent with your
observations? If a scenario is not consistent, briefly explain why (i.e. what observation
would be expected if that scenario were true?)
Scenario I : The pGLO gene is present on a DNA segment that does not contain a
promoter region.
Consistent with experimental observations?
Scenario II: The expression of the pGLO gene is controlled by a repressible arabinose
operon. Arabinose binds to a repressor protein causing the repressor to bind to a
promoter region that blocks the binding RNA polymerase.
Consistent with experimental observations?
Scenario III: The expression of the pGLO gene is controlled by an inducible arabinose
operon. Arabinose binds to a repressor protein locking the repressor in an inactive
conformation that prevents the repressor from binding to the promoter region.
Consistent with experimental observations?
Scenario IV: The expression of the pGLO gene is controlled by an operon. The arabinose
binds to an activator protein. When the arabinose is bound to the activator protein, the
protein binds to the promoter region and facilitates the binding of RNA polymerase,
thereby increasing transcription efficiency.
Consistent with experimental observations?
7) Thought Question: Which mechanism for pGLO gene regulation is correct Scenario
III or Scenario IV?
One technique for exploring DNA/protein complex interactions is called “Footprinting”.
DNA is exposed to DNAases in the presence of the suspected DNA binding proteins. If a
protein is bound, the region of DNA that is attached to the protein is protected from
degradation.
Figure I: No protein Bound to DNA:
DNA
+ DNAase
Figure II: Protein bound to DNA:
DNA Binding Protein
DNA
+ DNAase
You are a researcher trying to determine whether Scenario III or Scenario IV from
problem 6 is the more likely mechanism for regulating the gene expression of pGLO.
You perform the following experiments. First pGLO alone is digested by DNAase and
the fragments are separated by gel electrophoresis. Next, pGLO is incubated with
arabinose and then digested with DNAase and the DNA fragments separated by
electrophoresis.
The results are summarized below:
Experiment
Result
pGLO + DNAase
many fragments, promoter region digested
pGLO + DNAase + Arabinose fewer fragments, promoter region intact
Is this evidence more consistent with Scenario III or Scenario IV? Clearly explain
your reasoning.