Antibiotic Resistance:
pGLO™ Bacterial Transformation Lab
Objectives:
Students will describe antibiotic resistance as an example of evolution by natural selection.
Students will conduct an experiment to evaluate the spread of antibiotic resistance in cultured
bacteria.
BACKGROUND INFO:
In this lab, you will perform a procedure known as
bacterial transformation to introduce an
ampicillin-resistance (ampR) plasmid into the
bacteria Escherichia coli. This ampR plasmid
(named “pGLO”) contains the gene for betalactamase, an enzyme which provides resistance to
the antibiotic ampicillin, a member of the penicillin
family. The beta-lactamase protein is produced
and secreted by bacteria that contain the plasmid.
Instructor’s NOTES:
REMEMBER THE FOLLOWING SAFETY ISSUES ASSOCIATED WITH THIS LAB:
PENICILLIN ALLERGIES? (Ask ahead of time!!!)
Need to set up a BLEACH BUCKET for disposing of all bacterially contaminated items!!!
Goggles??
1. As the introduction to the lab, provide background information and instruct students to conduct research to identify and
define the bolded terms. See student instructions handout on the next page.
2. Once student lab groups have demonstrated their understanding of these terms and can explain them, move on to Day 1
activity – generation of E. coli starter plates. As from instructions on pages 16-17 on pGLO Kit instructions.
Appropriate questions to check understanding before student’s begin:
What is a plasmid? How are we going to get it inside the bacteria? What will happen if the plasmid is successfully
introduced inside the bacteria? What is beta lactamase? How does it inactivate ampicillin?
3. HW – DUE before performing transformation procedure: ANNOTATED Transformation Kit Quick Guide handout –
INSTRUCTIONS FOR HOW TO PERFORM THE TRANSFORMATION PROCEDURE.
Antibiotic Resistance:
pGLO™ Bacterial Transformation Lab
Objectives:
Students will describe antibiotic resistance as an example of evolution by natural selection.
Students will conduct an experiment to evaluate the spread of antibiotic resistance in cultured bacteria.
BACKGROUND INFO:
In this lab, you will perform a procedure known as genetic
transformation to introduce an ampicillin-resistance
(ampR) plasmid into the bacteria Escherichia coli. This ampR
plasmid (named “pGLO”) contains the gene for betalactamase, an enzyme which provides resistance to the
antibiotic ampicillin, a member of the penicillin family. The
beta-lactamase protein is produced and secreted by
bacteria that contain the plasmid.
PROCEDURE: To prepare for the lab, your group must:
A. Conduct research to EXPLAIN the experimental procedure we will be using, and to DEFINE and EXPLAIN
each of the terms below. For your research, go to our wikipage for Unit I, click the link to the “Bacterial
Transformation Lab OVERVIEW and TUTORIAL” and complete the tutorial. As you do so, take NOTES in
your Biochemistry Journal. In your notes, be sure to explain each of the following terms:
1.
2.
3.
4.
5.
6.
7.
Genetic transformation
Plasmid
ampR plasmid (“pGLO”)
Escherichia coli
Beta-lactamase
Enzyme
Ampicillin
B. Then WRITE, in PARAGRAPH FORM and using and EXPLAINING EACH of the 7 terms above, an
explanation of the Transformation Procedure we will use to generate bacteria that are able to grow in the
presence of the antibiotic ampicillin.
(HINT: Use the 6 step “Transformation Procedure” diagram of the tutorial to help you!).
C. Once you have completed the Tutorial, and written your paragraph explaining the transformation
procedure, check your understanding by completing the “Label the Results of Your Experiment” and “Lab
Quiz I” self-Quizzes at the end of the tutorial. What was your score?
DAY 1: Advance Preparation: Generation of E. coli starter LB plates. As students are conducting online
research/completing the online tutorial, teacher should circulate to different groups, demonstrating technique and
having each group generate their starter plate. See procedure below.
Antibiotic Resistance:
pGLO™ Bacterial Transformation Lab
Objectives:
Students will describe antibiotic resistance as an example of evolution by natural selection.
Students will conduct an experiment to evaluate the spread of antibiotic resistance in cultured bacteria.
Transformation of E. coli with the pGLO plasmid.
PROCEDURE: Before you begin the lab, your group must:
1. Have your HW: ANNOTATED Transformation Kit – Quick Guide
procedure handout on your desk.
2. Gather necessary Student Workstation Materials from the front of
the room. Use your group’s workstation Checklist to confirm that you
possess all of the required materials.
3. Review your Transformation Kit – Quick Guide by RE-READING THE
PROCEDURE so you remember what you are doing and when…
4. When your group thinks it is ready to begin, verify with Dr. Goldin that you may begin….
5. During logical pauses and breaks (e.g. during STEP 6, when your tubes are on ice for 10 minutes…),
record the following observations about the E. coli starter plate in your Biochemistry Journal:
a) The approximate total number of E. coli bacterial colonies on your LB plate
b) Distribution of the colonies on the plate. Is it even? Thicker in some places than others? Etc.
c) Color of the E. coli colonies as observed under normal light.
e) Color of the E. coli colonies as observed with an ultraviolet (UV) light
f) Ability of the E. coli bacterial cells to survive and reproduce in the presence of ampicillin
(see Dr. Goldin’s LB + Amp streaked plate for comparison to your LB plate).
Antibiotic Resistance: pGLO™ Bacterial Transformation Lab
STUDENT WORKSTATION MATERIALS CHECKLIST:
Quantity
Item












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E. coli starter plate (Your group’s plate you made yesterday)
Poured agar plates (1 LB. 2 LB/Amp, and 1 LB/Amp ara)
Plastic microfuge tube rack (to hold all your microfuge tubes)
Transformation Solution (in a small plastic microfuge tube marked “T”)
LB Nutrient broth (in a small plastic microfuge tube marked “LB”)
Empty Plastic microfuge tubes (use two different colors…)
Roll masking tape
Sterile bag of inoculation loops (you will use 7 of them…)
Sterile plastic transfer pipettes
Foam microfuge tube holder/float (for use on ice and in water bath)
Sharpie marking pen
UV light
Digital timer
Small tray of crushed ice.
1
4
1
1
1
2
1
1
5
1
1
1
1
1
COMMON WORKSTATION MATERIALS CHECKLIST:
 1
 1
 1
Rehydrated pGLO plasmid (ask Dr. Goldin…)
42◦C water bath with thermometer (back of room)
37◦C incubator with thermometer (back of room)
Antibiotic Resistance: pGLO™ Bacterial Transformation Lab
Objectives:
Students will describe antibiotic resistance as an example of evolution by natural selection.
Students will conduct an experiment to evaluate the spread of antibiotic resistance in cultured bacteria.
DAY 3: Observation of Results:
PROCEDURE:
The diagram below shows the 4 different bacterial transformation culture plates you made during our last class:
A. Before you get your plates, your group must answer the following questions in your Biochemistry Journal:
1. DRAW: In your Journal draw a schematic diagram for each of the four plates above.
2. EXPLAIN: Based upon the labels, what is the difference between these plates?
3. PREDICT: For each plate, what amount of bacterial growth do you expect to observe on each plate?
4. JUSTIFY your predictions based upon your knowledge of: the procedure; the process of genetic
transformation; and antibiotic resistance.
5. Which plate(s) is/are expected to contain genetically transformed E. coli bacterial cells?
6. Which plate(s) should be compared to verify that genetic transformation has occurred? EXPLAIN how you
can be sure that transformation has occurred.
7. Which plate is the “control” plate? What purpose does this plate serve? (Why do we need it?)
8. Once your group’s Recorder has verified that all group members have answered these questions, call Dr.
Goldin over to your bench.
B. Data Collection: Once you get your plates from Dr. Goldin, complete the following in your Journal:
1. Carefully observe and draw what you see on each of your four plates.
2. Compare your observations of the transformed “+ pGLO” E. coli and the non-transformed “- pGLO” E. coli.
3. Write down the following observations for each plate.
a) How much bacterial growth do you see on each plate, relatively speaking? If possible, count the total
number of bacterial colonies on each plate.
b) Which E. coli bacterial cells survive and reproduce in the presence of ampicillin (amp) and which do
not? WHY?
c) For each plate, what is the color of the E. coli colonies as observed under normal light?
d) For each plate, what is the color of the E. coli colonies as observed under ultraviolet (UV) light?
e) What is “ara”? How and why is the “ara” containing plate different? HINT: See d) above.
C. Data Analysis: Based upon the observations you made during data collection, as recorded in your Biochemistry
Journal, answer the following questions:
1. From the results that you obtained, how could you prove that the changes in bacterial growth that
occurred were due to the transformation procedure that you performed?
2. Do you observe some E. coli growing on the LB plate that does not contain ampicillin or arabinose? From
your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate?
Explain your answer.
3. How would you change the bacteria’s environment—the plate they are growing on—to best tell if they are
ampicillin resistant?