Bacterial Transformation with
Green Fluorescent Protein
pGLO Version
Table of Contents
Fall 2012
Bacterial Transformation
Introduction…………………………………………………………………………………………………………..1
Laboratory Exercise……………………………………………………………………………………….………..3
Important Laboratory Practices……………………………………………………………………………………3
pGLO Transformation Protocol………………………………...…………………………….....…………………4
Worksheet: Bacterial Transformation………………………………………………………………………..…....7
Acknowledgements………………………………………………………………………………….…………….10
Introduction to Bacterial Transformation
Student Version
Transformation is a process of transferring genetic information from one organism to another. In bacteria, a small
circular piece of DNA known as a plasmid (Table 1), transfers genetic information between bacteria, allowing these
microbes to gain antibiotic resistance and adapt to new environments.
This natural process can be modified by humans to increase our quality of life. In agriculture, genes are added to
help plants survive difficult climatic conditions, insect damage and increase their nutrients. Toxic chemical spills are
often controlled by transformed bacteria. Currently, many diabetics rely on insulin made from bacteria transformed
with the human insulin gene. Scientists use transformation as a tool to work on ways to treat other human diseases
and conditions.
Table 1: Illustration of a bacterial cell with chromosome and plasmids
Symbol
Bacterial structure
Illustration of E. coli
Plasmid containing a few genes
Circular bacterial chromosome
In this lab, you will be using non-pathogenic E. coli bacteria and pGLO, a plasmid modified with two genes. The
pGLO plasmid contains the genetic codes for (see Table 2):
1. a green fluorescent protein (GFP) from the bioluminescent jellyfish, Aequorea victoria
2. ampicillin resistance (amp)
3. a special gene regulation system (araC) requiring the carbohydrate arabinose to induce GFP expression
This means the GFP gene will only ‘turn on’ if arabinose is in the bacteria’s environment. If pGLO transformation is
successful and the bacteria are growing in arabinose, the colonies will appear neon green under UV light. These
fluorescing green bacteria must contain the pGLO plasmid with the GFP gene as well as the other genes found on
the pGLO plasmid. For this reason, the green fluorescent protein (GFP) gene is often used as a “reporter gene”
to identify expression of other genes of interest.
Table 2: pGLO plasmid and its three important genes.
Symbol
amp
araC
GFP
Type of gene
pGLO plasmid with inserted genes
Ampicillin resistance gene forming betalactamase, which inactivates ampicillin in
the media
araC gene creates a protein that binds
RNA polymerase to start transcription of
the GFP gene
GFP
araC
amp
GFP gene produces
the green fluorescent protein
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pGLO Transformation
Student Guide
Fall 2012
In order to successfully transform bacteria, you will need to add CaCl2, transformation solution (TS), to neutralize
both the bacterial cell wall and membrane charges, then, quickly shock them with a temperature change in order for
them to uptake the pGLO plasmid. After the stressful event, you will provide nutritious broth to restart their growth.
Deviating from the protocol listed below may decrease your success in obtaining transformants.
Will the untransformed bacteria appear neon green under a UV lamp? Will transformed bacteria fluoresce under a
UV lamp? List your predictions below. Before beginning the transformation, observe a plate of E. coli and a vial of
pGLO plasmid under a UV lamp. Then, view your transformed colonies once you complete the protocol below.
Explain your results.
Predictions & Actual Results
Item
View with
UV Lamp
Prediction
Explanation of Results
E. coli growing in petri dish
Vial of pGLO plasmid
Transformed E. coli in petri
dish
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pGLO Transformation
Student Guide
Fall 2012
Laboratory Exercise
The protocol outlined below describes a procedure for adding plasmid DNA to a bacterial cell. You will cool them
quickly and briefly heat the cells to move the plasmid DNA into the cells. Then, you will grow the cells on a petri
dish containing LB agar, antibiotics and arabinose. You will look for the development of green colored colonies of
bacteria.
Objectives - student should be able to:
1. Understand what a bacterial plasmid is and how it is used in biotechnology.
2. Understand how genes are used to make protein in a cell.
Im portant Laboratory Practices
a. Add reagents to the bottom of the reaction
tube, not to its side.
b. Add each additional reagent directly into
previously added reagent.
c. Do not pipet up and down to mix, as this
introduces error.
d. Make sure contents are all settled into the
bottom of the tube and not on the side or cap of
tube. A quick spin may be needed to bring
contents down.
Keep reagents on ice.
a. Pipet slowly to prevent contaminating the
pipette barrel.
b. Change pipette tips between each delivery.
c. Change the tip even if it is the same reagent
being delivered between tubes. Change tip
every time the pipette is used!
Check the box next to each step as you complete it.
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pGLO Transformation
Student Guide
Fall 2012
Place a check mark in the box as you complete each step.
pGLO Transform ation Protocol
1. Sterilize lab surfaces and wash hands before
beginning the lab.
2. Obtain two empty 1.5mL microfuge tubes from
your instructor. Using a permanent marker, label one
tube +DNA and the other tube –DNA. Label each tube
twice, on the lid and on the side.
Place these tubes into a Styrofoam cup containing
crushed ice.
2. Add 250µL of Transformation Solution (TS) to each
tube. If using a P-200 micropipettor, set the dial to
125µL and transfer 250µL (125µL, 2 times)
− DNA
+ DNA
!
!
250µL TS
Note:
TS contains calcium chloride (CaCl2), which helps
neutralize both the bacterial cell wall membrane and
DNA charges. Keep your tubes on ice.
!
4. Obtain a starter plate of E. coli. Observe the
colonies growing on it and note what you see.
UV#Light#
Wear safety glasses while using the UV lamp.
!
5. With a sterile inoculation loop, pick up one
bacterial colony from the starter plate.
Dip and swirl the loop into the +DNA tube to evenly
disperse the colony in the solution and release it from
the loop. With the cap closed, flick the tube with your
finger to mix.
Use a new loop to repeat the process for the -–DNA
tube. Return tubes to ice.
6. Wearing safety glasses, observe the contents of a
vial of pGLO under a UV lamp.
!
!
UV#Light#
!
Close the cap and flick the tube to mix the plasmid
with the contents of this tube.
Keep tubes on ice.
250µL pGLO
+ DNA
7. With a P-20 micropipettor, transfer 10µL of the
pGLO plasmid into your tube labeled +DNA only. DO
NOT add plasmid to the –DNA tube.
!
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pGLO Transformation
Student Guide
Fall 2012
− DNA
+ DNA
8. Incubate both tubes on ice for 10 minutes, making
sure the tubes are in contact with the ice.
!
10 minutes on ice
9. While you’re waiting, pick up these four plates:
1 LB, 2 LB/amp, 1 LB/amp/ara
Plate
LB
LB/amp
LB/amp
LB/amp/ara
On the outer edge on bottom of the plate, write +DNA
onto one LB/amp plate and the LB/amp/ara plate.
Write –DNA on the LB plate and the second LB/amp
plate. Also place your team initial or symbol on the
bottom of each plate.
+ DNA
Water&Bath&
42°C%/%50%seconds%
After 50 seconds, quickly place both tubes on ice for
another 2 minutes. It is VERY important to watch the
time and speed of the transfers.
2%min%
!
11. Return your tubes to a tube rack now resting on
your lab bench.
Add 250µL of LB broth to each of the tubes. If using a
P-200 micropipettor, set the dial to 125µL and transfer
250µL (125µL, 2 times)
− DNA
+ DNA
− DNA
+ DNA
− DNA
!
10. Heat shock your bacteria by transferring both
tubes to a foam rack and placing them into a water
bath set at 42°C for 50 seconds.
Make sure the tubes are pushed down as far as they
can go in the rack to contact the hot water.
Name
−DNA
−DNA
+DNA
+DNA
250µL LB
Remember to change the tips between the tubes!
!
12. Close the tubes. Mix each tube by flicking it
several times with your finger.
Incubate the tubes for at least 10 minutes at room
temperature. This process will allow the transformed
bacteria to recover and provide nutrients for their
growth.
10 minutes at room temperature
13. Obtain your four labeled plates. Transfer 100µL of
the appropriate solution to each labeled plate.
Remember to change tips between each transfer!
Plate
LB
LB/amp
LB/amp
LB/amp/ara
Name
−DNA
−DNA
+DNA
+DNA
Add100µL of cell solution
5
!
!
pGLO Transformation
Student Guide
Fall 2012
14. With a new sterile loop for each plate, spread the
liquid you just transferred onto each of the plates.
Evenly cover as much of the plate as possible.
Discard used tips into a waste container with
disinfectant. Allow bacteria to saturate into the agar
plate for a few minutes before the next step.
15. Invert your four plates. Then stack and tape them
together.
!
Place plates into an incubator oven set at 37°C until
the next day or when colonies are visible.
16. Decontaminate all lab surfaces with dilute
disinfectant and wash hands following the lab!
6
!
!
pGLO Transformation
Student Guide
Fall 2012
Name _________________________________________
Date __________________ Period__________________
W orksheet: Bacterial Transform ation
Lab Predictions
Will the untransformed bacteria, pGLO plasmid, and transformed bacteria all fluoresce green? Before viewing these
substances with a UV lamp, list your prediction on whether they will fluoresce green. Then, view them under a UV
lamp and provide an explanation of your results.
1. Predictions & Results
Item
Prediction
With UV lamp
Explanation
E. coli colony
Vial of pGLO
plasmid
Transformed E.
coli colony
2. Explain the purpose of these processes or substances during transformation.
Process
or
Purpose
Substance
a.
LB agar
b.
Prevents growth of untransformed bacteria on LB/amp plates.
c.
Calcium chloride
d.
Heat shock
e.
Arabinose
3. Describe 2 differences and 2 similarities between these Bacteria.
Condition
- pGLO DNA bacteria
+ pGLO DNA bacteria
Difference
Similarity
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pGLO Transformation
Student Guide
Fall 2012
Name _________________________________________
Date __________________ Period__________________
4. Before transforming your bacteria, list your predictions below for each of these petri dishes and their contents.
Then, describe your results following transformation.
Contents
LB -DNA
LB/amp -DNA
LB/amp +DNA
LB/amp/ara +DNA
Predictions*
Illustration of
Results
Description of
Results
*Possible responses for Predictions - Growth or no growth, fluorescence or no fluorescence under UV light, number
of colonies, etc.)
5. Compare your predictions with your actual lab results. Describe how close your predictions were to your actual
results and explain possible reasons for any differences.
6. Explain what may have occurred to produce these results. ( • = colony)
Contents
LB -DNA
LB/amp -DNA
LB/amp/ara +DNA
Illustration of
Results
Description
of Results
Possible
explanation
for results
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pGLO Transformation
Student Guide
Fall 2012
Name _________________________________________
Date __________________ Period__________________
7. If growth appeared on the LB/amp +DNA plate, would these bacteria…
a. be transformed? Explain.
b. fluoresce under UV light? Why or why not?
8. Provide an example of how transformation can be beneficial and an example of how it can be potentially harmful
to humans.
Condition
Transformation example
a.
Beneficial
b.
Harmful
9. Although transformed cells appear white, with the same phenotypic expression of the wild-type bacteria when
the growth media lacks arabinose, they will fluoresce green with a long-wave UV lamp when arabinose is
present. Explain why this color change occurs.
10. Provide a rational or benefit of adding DNA sequences coding for fluorescent proteins such as GFP, to tag
genes of interest in plasmids used for transformation.
11. In your own words, explain the process of transformation.
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pGLO Transformation
Student Guide
Fall 2012
BABEC Educational Transform ation Kits
For Research Use Only. Not for use in diagnostic procedures.
BABEC thanks Qiagen for their generous support of plasmid prep kits for BABEC bacterial transformation labs.
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pGLO Transformation
Student Guide
Fall 2012