AP Biology 12
AP Lab Invertigation 8 – Biotechnology: Bacterial Transformation
How can we use genetic engineering techniques to manipulate heritable information?
Glowing Bacteria: Transformation of the bacteriaum Escherichia coli (E. Coli) with a Jelly Fish Gene
***The following lab uses a procedure adapted from BioRad***
Overview
Plasmids containing specific fragments of foreign DNA will be used to transform Escheruchia coli cells, passing
on antibiotic (ampicillin resistance) and foreign gene (jellyfish GFP – green fluorescen protein) expression.
Learning Objectives
1)
2)
3)
4)
To demonstrate the universality of DNA and its expression
To explore the concept of phenotypic expression in organisms
To explore how genetic information can be transferred from one organism to another
To investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in
organisms
5) To explore the relationship between environmental factors and gene expression
6) To investigate the connection between the regulation of gene expression and observed differences
between individuals in a population of organisms.
Procedure
You will insert the plasmid pGlo into competent E. coli cells. The pGlo plasmid has: the GFP gene, which is
turned on by exposure to arabinose (ara), and ampicillin (amp) resistance. Before coming to the lab, be sure to
complete all the predictions.
Note: Note that all volumes must be absolutely precise. There will be no waste for any of the solutions/broths.
If you use too much, you will run out. Also note the sterile equipment. Contamination is a risk, so you must
follow the procedure with diligence. Ms. Wood will be placing you into eight lab teams. Before starting the lab,
you will have a team meeting to compare your pre-labs and decide on “jobs.” Ensure you have completed all
predictions.
***The bacterium Escherichia coli, strain HB101;K-12, is made of only one cell, reporoduces every 20 minutes,
it does not make people sick, and it cannot survive outside the laboratory.***
AP Biology 12 – Investigation 8 – Transformation
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No UV lamp on Day 1
AP Biology 12 – Investigation 8 – Transformation
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Eight plates NOT four – Why did Ms. Wood Change to Eight Plates?
-
LB/ara
+
+
LB/ara
Steps 10-12: Make appropriate
adaptations to accommodate for
your eight plates
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Day 2
1) Remove your plates from the incubator.
2) Count the number of colonies growing on each plate and record the value in the results; use a
permanent marker to mark each colony on the bottom of the plate (not the lid) as it is counted. If the
cell growth is too dense to count individual colonies, record “lawn.” Do not open the plates for counting!
3) Take into a dark area (W203A) and observe any glowing under UV light! Wear the UV goggles.
4) Compare results to predictions.
5) What does each plate tell you about the experiment?
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Table 1. Predictions
Plate
Predictions
Colony Growth?
#colonies/lawn/none
Glowing? Yes/No
LB+
LBLB/Amp+
LB/AmpLB/Ara+
LB/AraLB/Amp/Ara+
LB/Amp/Ara-
Table 2. Results
Plate
Actual Results
Colony Growth?
#colonies/lawn/none
Glowing? Yes/No
LB+
LBLB/Amp+
LB/AmpLB/Ara+
LB/AraLB/Amp/Ara+
LB/Amp/AraOther Observations and Experimental Notes:
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Analysis of Results
What does each plate tell you about the experiment?
How do your results compare to your predictions?
How do your results compare with other lab teams?
What is the significance of what you just did?
Conclusion
Refer to the criteria on the lab rubric.
Beginning
Developing
Accomplished
Exemplary
o
Point form
o
Poor sentence structure
(fragments)
o
Full sentences
o
o
Purpose and/or results not
referred to
o
Refers to purpose, summarizes
results without insight
o
Answers purpose by
summarizing results
o
o
No sources of error
o
Sources of error not relevant
o
States 1-2 relevant sources of
error
o
o
No connection to relevance
o
Relevance attempted, but
invalid
o
Reveals relevance of results
o
o
Clear communication through
structured sentences that
connect one idea to the next
Answers purpose by comparing
key results to
predictions/hypothesis
(supported/
rejected/inconclusive)
States 2 or more insightful
sources of error and suggests
improvement to this lab
Connects to big picture
(relevance)
Asks new questions and
suggests new experiments for
the future
Further comments/suggestions:
(To make sure you’ve understood the concepts of this experiment, answer questions #1-4 on page 310 in
Holtzclaw.)
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