Introduction to Genetic Engineering
Bacterial Transformation with
Green Fluorescent Protein (pGLO)
Table of Contents
Bacterial Transformation Lab Activity
Introduction ……………………………...…………………………………………………….…………………….1
Background Information and Scientific Theory ………………………………………………………….………2
General Lab Skills Required ………………………………………………………………………………………4
Laboratory Activity …………………………..……………………………………………………….……….…….6
Worksheet: Bacterial Transformation ……………….………………………………..……………...……..…....9
Worksheet: Calculating Transformation Efficiency ………………………..…………………………………..12
Appendix for Student Guide …………………………………………………………………..…………………15
Acknowledgements………………………………………………………………………………….…………….19
www.babec.org
Introduction to Genetic Engineering
Bacterial Transformation with
Green Fluorescent Protein (pGLO)
Genetic engineering is an umbrella term that encompasses many different techniques for moving DNA between
different organisms. Transformation the process by which an organism acquires and expresses a whole new
gene. In this activity, you will have the opportunity to genetically transform bacteria cells; altering them so that they
can make an entirely new protein. This procedure is used widely in biotechnology laboratories all over the world,
enabling scientists to manipulate and study genes and proteins in exciting new ways.
Adding a new gene to bacteria cells is a very simple procedure. You will add a gene that codes for Green
Fluorescent Protein (GFP). This protein was discovered in the bioluminescent jellyfish called Aequorea victoria, a
jellyfish that fluoresces and glows in the dark (Figure 1).
The gene for GFP was isolated in 1994 and was quickly used in laboratories as a way to brightly label proteins in a
living cell. This “tagging” of proteins allowed researchers to visualize specific proteins to learn more about their
biological functions in exciting new ways. The discovery of GFP proved to be so important that the Nobel Prize in
Chemistry in was awarded to Osamu Shimomura, Marty Chalfie and Roger Tsien in 2008 for their work. Since
then, Roger Tsien’s laboratory at UCSD has altered the GFP gene to make a full rainbow of proteins. Figure 2
shows how bacterial expressing many different colored fluorescent proteins can be grown together on one plate.
Figure 1
Aequorea victoria
glowing under UV light
Figure 2
A rainbow of fluorescent
growing on an agar plate
Bacteria are commonly used for genetic transformation experiments because they are simple, single-celled
organisms that grow and reproduce very quickly. Bacteria cells store their DNA on one large, circular chromosome.
But they may also contain one or more small circular pieces of DNA called plasmids. Because bacteria reproduce
asexually, plasmid DNA allows for the addition of new traits into a cell. Plasmids are able to replicate independently
of the large bacterial chromosome, and can transfer easily between cells. Figure 3 shows the circular DNA
chromosome and plasmid DNA inside of a cell.
Figure 3
Genetic material in bacteria
takes 2 forms
Bacterial evolution and adaptation in the wild often occur via plasmid transfers from one bacterium to another. An
example of bacterial adaptation is resistance to antibiotics via the transmission of plasmids. 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 or damage from insects, and to increase their absorption of nutrients. Toxic chemical
spills can often be bioremediated (cleaned-up) by transformed bacteria specifically engineered to do the task.
Currently, many people with diabetes rely on insulin made from bacteria transformed with the human insulin gene.
Scientists use transformation as a tool to study and manipulate genes all the time.
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Background Information and Scientific Theory
The Central Dogma of Molecular Biology
A basic tenet of biology, from single-celled bacteria to eukaryotes, is the mechanism of coding, reading and
expressing genes. The central dogma of molecular biology states that: DNA > RNA > PROTEIN > TRAIT. This
curriculum is an example of the central dogma in action. The instructions for GFP production are encoded in the
DNA. When transcription is turned on, the cell turns those instructions into an mRNA transcript. This transcript is
then translated into protein, which provides the trait of fluorescence.
Gene Regulation
Every cell in the human body shares an identical genome that contains over 20,000 different genes. But if all cells
have the same genes, how is it that a muscle cell ends up being so very different from a brain cell? The answer
lies in the fact that there is a specific process for controlling which genes are turned “on” and which are turned “off”
in every single cell. Gene regulation is the name for all the different cellular processes that have to take place in
order for a gene to be turned into a protein.
Gene regulation is an important concept in biology dictating where and when genes are turned on or off. Gene
expression occurs when genes are turned on, resulting in the expression of proteins – the workhorses of the cell.
Proteins called transcription factors are frequently used by cells to turn transcription on or off depending on
environmental conditions. They are important for cellular development, tissue specialization, and organismal
adaptation to the environment. Transcription factors act at the promoter region in front of a gene. At the promoter,
RNA polymerase initiates transcription and turns a gene on; the gene is then said to be “expressed”. Once the
mRNA transcript is made, it can be translated into protein. All the genes in our bodies are highly regulated to allow
for maximum efficiency, and to decrease waste (energy) in our cells.
The pGLO System
In this laboratory activity, you will have the opportunity to genetically engineer a cell and you will see with your own
eyes the critical role of gene regulation in living systems. This is because the expression of the GFP gene in this
experiment is not automatic. Rather, it happens only when the environmental conditions are just right.
Plasmids used by molecular biologists are named with an acronym that begins with the lower case "p", and
followed by a name that conveys information about its function. pGLO is the name for a plasmid that has been
engineered to contain the gene for GFP, which glows under UV light. Using recombinant DNA technology,
scientists designed this plasmid to contain two additional genes, for a total of three genes whose function it is
important to understand before beginning this activity.
Figure 4: The pGLO plasmid has been engineered to express 3 genes
ara
ara
Codes for the regulatory protein araC, which works with the sugar
arabinose to turn on GFP transcription by recruiting RNA polymerase
GFP
Codes for Green fluorescent protein, which is derived from Aequorea
victoria - a bioluminescent jellyfish that fluoresces under UV light.
amp
ampr
r
Codes for the enzyme beta-lactamase, which inactivates the
ampicillin and allows the cell go grow in the presence of an antibiotic.
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In this lab activity, you will be inserting pGLO into non-pathogenic E. coli bacteria. The procedure is never 100%
efficient and only a few of your E. coli bacteria will successfully “take up” the pGLO. How will you know which cells
contain the plasmid? pGLO contains a gene that codes for a protein that protects the cell against the toxic effects
of antibiotics. This means that only cells that have the plasmid will survive in the presence of antibiotics. In this
procedure, we use ampicillin, an antibiotic very similar to penicillin. This step is called antibiotic selection, and it
allows you to select only the cells that have been transformed. The beta-lactamase gene in pGLO codes for a
protein that breaks down ampicillin. Expression of the beta-lactamase gene in cells that have been successfully
transformed allows them to grow in the presence of ampicillin. Non-transformed cells will die.
Your transformed cells will grow on a plate with ampicillin, but they will not fluoresce green until the GFP gene is
turned “on”. Here’s where the idea of gene regulation comes into play. Transformed cells will grow on plates not
containing arabinose, but will only fluoresce green under UV light when arabinose is included in the nutrient agar.
Therefore, arabinose, a sugar that bacteria consume for energy, is the critical ingredient for making your bacteria
glow.
What’s so special about arabinose? It teams up with the araC, the regulatory protein that is expressed by pGLO.
Regulatory proteins control the timing and location of many cellular processes. Specifically, araC is a transcription
factor which, as described previously, functions to turn genes on and off. But it can’t turn GFP on by itself – it
needs the help of arabinose. Together, they work to bring in RNA polymerase, the enzyme that makes RNA, and
only then can the glowing, green protein be made. It's a finely orchestrated dance, and all the right players have to
be in place for success.
Figure 5
Gene regulation of
GFP in pGLO
Figure 5 shows that when araC teams up with arabinose, its shape changes. The protein araC easily forms a bond
with the sugar arabinose, and only when they both get close together can the complex function as a transcription
factor. What it then does is very simple: it stimulates RNA polymerase to start transcription, and we see firsthand
the central dogma of molecular biology in action!
The Transformation Procedure
In order to increase the chances that your E. coli will incorporate foreign DNA, you will need to alter their cell
membranes to make them more permeable. This is a two-step process. First you place your cells and pGLO
together in a transformation solution (which contains calcium chloride) to neutralize the charge. Second, you
o
quickly heat shock them with a temperature change (42 C). This hot temperature permeabilizes (loosens) the
bacterial cell wall, making it easier for pGLO to cross it. This process can be harmful to the cells, so you want to
give them a nutritious broth to restart their growth as soon as you’re done. Luria Broth (LB) is a liquid that contains
proteins, carbohydrates and vitamins so that the E.Coli can rapidly recover and thrive. They will then be placed on
an agar medium, a jello-like substance containing LB, to grow overnight.
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General Lab Skills Required for Success
Using Sterile Technique
Students should wash their hands before starting lab, after handling recombinant DNA organisms/containers, and
before leaving the lab area. All lab surfaces should be decontaminated at least once a day during each class
section and following spills. Students should avoid touching the tips of the pipettes or inoculating loops onto any
contaminating surfaces, unless instructed in the protocols. Students should practice proper aseptic techniques to
prevent contamination.
Using Microipettes
Students need to be familiar with micropipetting techniques and remember to exchange pipet tips to avoid cross
contamination. Do not carry micropipettes sideways or upside down while transferring liquids. Please don’t abuse
the micropipettors by dialing in amounts beyond their intended calibration limits. When transferring liquids, the
student holding the micropipettor should also be holding the microfuge tube of liquid to transfer. Both should be
brought to eye level in order to visually confirm that liquid has been transferred. A teammate should confirm the
correct micropipettor setting, correct tube of liquid to transfer and the use of clean pipet tips. Success of the lab
depends on the proper use of tools and reagents required for the protocol.
UV Safety
Ultraviolet radiation can cause damage to eyes and skin. Use UV-rated safety glasses or goggles if looking directly
at UV light.
Using Experimental Controls
In this lab, it is important to confirm which cells have received the plasmid, and under which conditions the green
fluorescent proteins are being produced. You will need to prepare a series of experimental controls to be able to
interpret your results correctly. These controls are designed to minimize the effects of factors other than the single
concept that you are testing. Therefore, 2 different reactions will be performed: one with pGLO plasmid (+pGLO)
and one without it (- pGLO). See Figure 6 to understand how to set up your reactions.
Figure 6: Bacterial growth conditions
#1
#2
#3
Nutrient
Agar (LB)
Antibiotics
(ampicillin)
The - pGLO control serves two roles: 1) to ensure that the
bacteria are still alive after the chemical and heat shock
procedure, and 2) to make sure that the ampicillin is
working property. You will plate these bacteria under
conditions #1 and #2, but you should only expect them to
grow in condition #1.
The +pGLO transformation will grow in condition #1 and
#2. However, only the bacteria that successfully took up
the pGLO plasmid will grow in condition #2. In this
reaction, you will observe the process of antibiotic
selection, but you should not see any GFP produced.
“On”
Switch
(arabinose)
E.Coli
+ pGLO
Yes
Yes
Yes
E.Coli
- pGLO
Yes
Yes
No
No
Condition #3 is only used for the +pGLO reaction. This
example proves that the transcriptional control of the GFP
gene is intact. The bacteria on this plate are the only ones
that should glow green when exposed to UV light. In this
reaction, you will observe the process of gene regulation.
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The protocol outlined next describes the procedure for adding plasmid DNA to a bacterial cell. Be sure to follow
each step very carefully. You will be cooling your E. Coli cells on ice, then heating them in a water bath, then letting
them recover. Make sure your pipetting volumes are accurate at every step. Afterwards, you will grow the cells on a
petri dish containing LB agar, antibiotics and arabinose. After 1-2 days, you will look for the development of green
fluorescent colonies of bacteria.
Student Learning Outcomes – at the end of this laboratory, students will be able to:
1. Describe the central dogma of molecular biology.
2. Explain the process of bacterial transformation and selection.
3. Relate the use of bacterial transformation in biotechnology.
4. Differentiate transformed from non-transformed cells.
5. Calculate transformation efficiency and compare with the class data.
Preliminary predictions and questions to think about
Will the untransformed bacteria appear neon green under a UV lamp? Why or why not?
Why don’t you attempt to grow the –pGLO reaction under growing condition #3 (page 4)?
Why are there so many fewer bacterial colonies for the +pGLO reaction under condition #1 than condition #2?
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 lab activity. Do you see glowing? Fill out the table below:
Item
View with
UV Lamp
Prediction
Explanation of Results
E. coli growing in petri dish
on LB agar
Vial of pGLO plasmid
Transformed E. coli grown
under condition #2
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Laboratory Activity
Place a check mark in the box as you complete each step.
pGLO Transformation Protocol
1. Sterilize lab surfaces and wash hands before
beginning the lab.
2. Obtain one empty 1.5mL microfuge tubes from
your instructor. Using a permanent marker, label the
tube +DNA.
Negative Control:
Assigned groups will perform a mock
transformation to be used as negative control for
the class. Label a second tube –DNA.
Label each tube twice, on the lid and on the side.
Place these tubes into a Styrofoam cup containing
crushed ice.
3. Add 250µL of Transformation Solution (TS) to each
tube using a proper micropipette (Alternatively you
can use a plastic transfer pipette)
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.
Place the plate on the UV lamp and observe the
colonies. Are they glowing?
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.
If doing the negative control, 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. Does it glow?
UV Light
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7. With a P-20 micropipettor, transfer 10µL of the
pGLO plasmid into your tube labeled +DNA only.
(Alternatively, use the 10µL inoculation loop. Dip the
loop into the 2mL stock plasmid tube. A noticeable
film will form around the ring due to surface tension
(like a bubble wand). Swirl the loop into tube labelled
+DNA.)
+ DNA
10µL pGLO
**DO NOT add plasmid to the –DNA tube.
Close the cap and flick the tube to mix.
− 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 3 plates:
1 LB, 1 LB/amp, 1 LB/amp/ara
On the outer edge on bottom of the plate, write +DNA.
PGlo
Transformation
+DNA
LB
LB/amp
LB/amp/ara
If performing the negative control experiment, pick up
1 LB and 1LB/Amp plate. Label them –DNA.
Negative
control
-DNA
LB
LB/Amp
− DNA
+ DNA
Make sure the tubes are pushed down as far as they
can go in the rack to contact the hot water.
− 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.
− DNA
Also write your team initial or symbol and the date on
the bottom of each plate.
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.
Using a proper micropipette (or transfer pipette) , add
250µL of LB broth to each of the tubes
250µL LB
Remember to change the tips between the tubes!
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12. Close the tubes. Mix each tube by flicking it
several times with your finger.
Incubate the tubes for at least 10-20 minutes at 37°C.
You can use the bacterial incubator or other warm
place like the top of a refrigerator or keeping the tube
warm in your hands for this step. This process will
allow the transformed bacteria to recover by providing
nutrients for their growth.
10-20 minutes at 37°C
13. Obtain your labeled plates. Using a p200 (or
sterile transfer pipette), transfer 150µL of the +DNA to
each plate labeled +DNA plate.
Be careful not to poke into the agar!
PGlo
Transformation
+DNA
LB
LB/amp
LB/amp/ara
14. Using a clean inoculation loop, gently spread the
liquid on the agar of each plate. You may use the
same loop for all the +DNA plates.
Be careful not to poke into the agar!
Evenly cover as much of the plate as possible.
Discard used loops into a waste container with
disinfectant. Allow bacteria to saturate into the agar
plate for a few minutes before the next step.
15. If performing the negative control experiment,
repeat step 13 and 14 using the -DNA on the
appropriately labeled -DNA plates.
Negative control
-DNA
LB
LB/Amp
Use a clean transfer pipet and inoculation loop for this
set.
16. Invert your 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.
Alternatively, stack the plates in a warm spot in the
classroom. It may take 2-3 days for bacterial colonies
to appear.
After the colonies have appeared, you may keep the
plates by wrapping them in parafilm and storing in the
refrigerator
17. Decontaminate all lab surfaces with dilute
disinfectant and wash hands following the lab!
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Name ________________________________________
Date __________________ Period__________________
Worksheet: Bacterial Transformation
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.
Ampicillin or
antibiotic
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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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, +DNA
LB/amp, +DNA
LB/amp/ara, +DNA
Predictions*
Illustration of
Results
Description of
Results
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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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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Worksheet: Calculating Transformation Efficiency
When performing transformation experiments, you usually want to obtain as many transformants as possible. This
is important because you want to make sure your conditions for transformation is at its optimum. Transformation
efficiency is the efficiency whereby cells take up the introduced DNA. Many factors contribute to transformation
efficiency: cell age and competency (the ability to take up DNA), the type of cells being transformed, plasmid length
and quality, the method of transformation (heat shock or electroporation) and just different conditions in general.
Having a low transformation efficiency may point to poorly competent cells, poor conditions, or poor techniques (not
following protocol). In a research lab, it’s good to have many transformants for research, just in case individual
transformants may not work as well (e.g. different levels of expression), or some other unknown problems
associated with transformed cells. In making a genomic library, you want as many transformants as possible to
have a robust library. In cell culture, you may take a population of transformed cells for further study therefore
having a high transformation efficiency allows for better study.
In this exercise, we will calculate the transformation efficiency of the E. coli bacteria by pGLO . The data can then
be gathered from each team of the class and the data compared with a different transformation technique called
electroporation.
Transformation efficiency calculation: The number of colonies observed growing on an agar plate (cfu)
Amount of DNA used (in µg)
cfu=colony forming units
Two data are needed for this:
1. Total number of green fluorescent colonies on your LB/amp/ara plate.
2. Total amount of pGLO plasmid DNA used for bacterial transformation that was spread on the LB/amp/ara
plate.
1. Determine the total number of transformed green fluorescent colonies.
Place the LB/amp/ara plate near a UV light source. Count the number of green fluorescent colonies that
glow under UV light.
Enter that number here
Total number of colonies = _____________
2. Determine the amount of pGLO DNA in the cells spread on the LB/AMP/Ara plate.
Two pieces of information are needed:
a) The total amount of DNA you used for the +DNA in the experiment
b) The fraction of DNA that was spread onto the LB/amp/ara plate
a. Total amount of DNA:
DNA in mg = (concentration of DNA in µg/µ
µl) x (volume of DNA in µl)
In this experiment, 10µl of pGLO at a concentration of 0.01
Enter that number here
µg /µl was used.
Total amount of pGLO DNA,
µg used in this experiment = _____________
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b. Fraction of pGLO plasmid DNA (in the bacteria). For this experiment, a certain amount was spread onto
each plate. To find that fraction:
Sample volume spread on LB/amp/ara plate, in µl
Total sample volume in tube, in µl
Fraction of DNA used
•
150µ
µl of cells was spread from the tube containing a total volume of 500µ
µl of solution.
Enter that number here
c.
Fraction of DNA= _____________
How many µg of pGLO DNA was spread on the LB/amp/ara plate? Multiply the total amount of pGLO DNA
used by the fraction of pGLO DNA you spread on the LB/amp/ara plate.
pGLO DNA spread (µg) = amount of DNA used (µg) x fraction of DNA
Enter that number here
pGLO DNA spread, µg = _____________
Now, we are finally ready to calculate the transformation efficiency!
Number of colonies on LB/amp/ara plate = _______________
pGLO DNA spread, µg = _____________
Transformation efficiency calculation: The number of colonies observed growing on an agar plate
Amount of DNA used (in µg)
Enter that number here
Transformation efficiency = _____________ transformants or
cfu/µ
µg
cfu=colony forming units
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Analysis of results: What is the transformation efficiency of each team in the class?
Team
Efficiency
In past studies, this method of “heat shock” protocol that was performed by research labs usually has a
2
3
transformation efficiency between 8x10 and 7x10 transformants per microgram of DNA.
How does your team’s result compare to this data?
How does the class’ result compare to your data and to the data by research labs?
Another method for transformation is called electroporation. In this method, an electric field is applied to allow the
8
cell membrane to open up and take up DNA. The transformation efficiency from electroporation may be 1x10
cfu/µg. What fold higher is the transformation efficiency by electroporation vs. heat shock?
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Appendix for Student Guide
Streaking starter plates of E. coli
Starter plates are needed to produce bacterial colonies of E. coli on agar plates. LB agar plates should be streaked
to produce single colonies and incubated at 37°C fo r 24–36 hours before the transformation investigation begins.
Under favorable conditions, one cell multiplies to become millions of genetically identical cells in just 24 hours.
There will be millions of individual bacteria in a single millimeter of a bacterial colony.
Depending on time, you may prefer your students to learn how to streak their own plates for individual colonies.
Plate Streaking
Streaking takes place sequentially in four sections.
The first streak spreads out the cells. In subsequent
streaks the cells become more and more dilute, thus
increasing the likelihood of producing single colonies.
1. Using a sterile inoculation loop, pick up a
bacterial colony from live E. coli culture plate.
Using a back and forth motion, gently spread
the colony into one quadrant of the LB starter
plate. Keep the lid slightly tilted open - only as
much as necessary. Be careful not to
puncture the agar.
2. Rotate the plate one-quarter of a turn. Go into
the previous streak about two times and then
back and forth as shown for a total of about
10 times.
3. Again, rotate the plate one-quarter of a turn
and pass over a previous streak from the
previous quadrant several times with the loop.
4. Repeat step 3, but this time, drag out the loop to form a tail not touching any previous streaks Close your
plate to avoid further contamination.
5. Place the used loop in a disinfectant solution waste cup. Follow this procedure for the remaining starter
plates. Once starter plates are inoculated, incubate them upside down in a 37°C incubator oven for 24 to
36 hrs.
6. If your students are not using the plates right away, seal the sides with Parafilm or lab tape so they don’t
dry out, invert the plates, and place them in a dark cupboard until needed. Avoid refrigerating your starter
plates as cooling will reduce your transformation efficiency.
What to expect the next day
You should see individual bacterial colonies in quadrant 4, and very dense bacterial growth in quadrant 1.
Quadrants 2 and 3 will have bacterial density somewhere in between, similar to what is seen below:
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Your streaked plate should look similar to this image after 24 – 36 hours:
Note: the images on this page have been provided by the Florida Institute of Technology
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Important Laboratory Concepts Covered
Lab Safety and Disposal
If you do not have an autoclave readily available, all solutions and equipment coming into contact with the bacteria
such as the pipet tips and inoculating loops, should be collected and placed into disinfectant solution, such as 10%
bleach. Cover each contaminated petri dish with disinfectant solution and let sit for at least 15min before disposal
according to your site guidelines. For your protection, wear safety glasses and a lab coat when handling
concentrated and dilute disinfectant. Make sure there is adequate ventilation.
Your school’s specific safety and disposal policies should always take precedence.
The E.coli used in this lab
E.coli is a bacteria found everywhere in our environment. The strain we use for this lab and in many research
scientific labs are harmless to humans and are NOT pathogenic. They have been specially engineered to help
scientist with their work. If you touch the bacteria with your hands, simply wash with soap and water. If you get
some bacteria in your eyes, simply flush with water. As always, use safety precautions when working in the
laboratory.
Media and Additives
LB (Luria-Bertani) agar and broth contain a yeast extract with a mixture of amino acids, carbohydrates, salts and
vitamins. Together, these substances support bacterial growth. Agar contains a gel derived from seaweed that
solidifies at room temperature. If you have extra prepared dishes, allow your students to touch the surface of one of
the LB plates and help them make connections between agar and Jello.
Including the antibiotic ampicillin in the media prevents the growth of bacteria other than the successful
transformants. The pGLO plasmid contains a beta-lactamase gene, which allows the transformed bacteria to
produce an ampicillin inactivating protein, the enzyme beta-lactamase. Using ampicillin in the media insures that
only bacterium containing and producing beta-lactamase will grow. Seeking survival of only the transformed cells is
an example of antibiotic selection. Ampicillin breaks down over time and is sensitive to heat and repeated
freeze/thaw cycles.
The addition of the carbohydrate sugar arabinose in the media will activate the transcription of the GFP gene.
Translation can then follow, resulting in expression of the GFP protein. The GFP protein allows the transformed
cells to appear neon green with a long-wave UV lamp or standard UV transilluminator. Without arabinose in the
media, the GFP gene will not be transcribed, and the GFP protein will not be produced. The phenotypic expression
of the “wild-type” bacteria is white. Transformed cells will also appear white when the growth media lacks
arabinose, but they will fluoresce green under UV light, if arabinose is present. This engineered pGLO plasmid
allows students and teachers to easily verify their transformation success.
Try this: If you have extra arabinose (and the time), add at least 100µl of the arabinose onto a previously
inoculated “LB/amp +DNA” plate. Cover, wait a couple of minutes to allow the arabinose liquid to soak into the agar,
invert, and incubate this plate for 18-30 hrs to demonstrate how the GFP gene can be “switched-on” by the new
presence of arabinose in its environment.
Transformation Solution (TS), 50mM CaCl2
When fully intact, the bacterial cell membrane does not allow DNA to pass through it, so how do we get the DNA
2+
inside during transformation? We add Ca cations, which neutralize the negative charges of both the DNA
phosphate-backbone and the phospholipids within the cell membrane. By neutralizing these repulsive negative
charges, the DNA can then easily pass across the bacterial cell membrane. It is possible to get transformants if
CaCl2 is missing. However, the efficiency (number of colonies on plates) might be very low. A great animation of the
process is available at http://www.dnai.org > Manipulation > Techniques > Transferring & Storing > Transformation
Animation.
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Heat Shock
Heat shock helps bacterial cells take in small foreign DNA segments such as plasmids by increasing a cell
membrane’s permeability. Students must carefully follow the pre-optimized process laid out in this protocol; it
contains specific temperatures and incubation times that will ensure success. Otherwise, few, if any, bacteria will
uptake the plasmid and be transformed. A great animation of the process is available at http://www.dnai.org >
Manipulation > Techniques > Transferring & Storing > Transformation Animation.
Recovery
The 10min incubation period in nutrient LB broth after the stress of heat shock allows the transformed bacteria cells
to heal and grow. They will also begin to secrete beta-lactamase, the ampicillin inactivation enzyme, which
increases the survival rates of the transformed cells on the ampicillin plates.
Incubation
Optimal growth for E.coli occurs at 37°C. E. coli will require more time to grow and express the GFP gene if kept at
room temperature. A warm spot on top of the refrigerator or heating units in the classroom will help. It will take 2-3
times longer for the bacteria to grow at room temperature versus an incubator set at 37°C, but they wi ll grow in
these conditions.
Antibiotic Selection
The pGLO plasmid, which contains the GFP gene, also contains the gene for beta-lactamase. Beta-lactimase is an
enzyme that 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.
Beta-lactamase inactivates the ampicillin present in the LB nutrient agar to allow bacterial growth. Only transformed
bacteria that contain the plasmid and express beta-lactamase can grow on plates that contain ampicillin. Only a
very small percentage of the cells successfully take up the plasmid DNA during heat shock and are transformed.
Untransformed cells cannot grow on the ampicillin selection plates.
In order to "stably retain" the plasmid, there needs to be some type of metabolic reason for the E. coli to keep the
plasmid around. If the plasmid contains a gene that codes for a protein that protects against antibiotics, then only
cells that have the plasmid will survive in the presence of that antibiotic
Aseptic technique
When growing bacteria in culture, it is important to prevent the growth of unwanted microorganisms in the nutrient
rich media. Aseptic technique is a series of methods that are used to minimize the chances of contamination.
Examples include use of sterile tubes and pipettes, sterilized solutions, cleaning the work area with disinfectants,
use of Bunsen burners, and keeping the caps of tubes, plates and pipette boxes closed.
Using student workstations
It is recommended that students work in groups of two. It is up to the discretion of the teacher as to whether
students should access common buffer solutions and equipment or whether the teacher should aliquot solutions in
the microtubes provided.
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BABEC Educational Transformation Kits
www.babec.org
BABEC thanks Qiagen for their generous support of plasmid prep kits for BABEC bacterial transformation labs.
Acknowledgements
The following images have been provided courtesy of:
Figure 1 – adapted from National Geographic. http://voices.nationalgeographic.com/2012/04/03/love-and-war-theessence-of-luminosity/
Figure 2 – Tsien Laboratory at UCSD. http://www.tsienlab.ucsd.edu/Images.htm
Figure 3 – Wikipedia. https://en.wikipedia.org/wiki/Plasmid
Figure 4 – adapted from Bio-Rad. www.bio-rad.com
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Bacterial Transformation: Student Version
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