Lab 4 and 5: pGLO Transformation

Introduction to Transformation

In this lab you will perform a procedure known as genetic transformation. Remember that a
gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein
gives an organism a particular trait. Genetic transformation literally means “change caused by genes,”
and involves the insertion of a gene into an organism in order to change the organism’s trait. Genetic
transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as
frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria
can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases
caused by defective genes are beginning to be treated by gene therapy; that is, by genetically
transforming a sick person’s cells with healthy copies of the defective gene that causes the disease.

You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent
Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Green
Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Following the transformation
procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein,
which causes them to glow a brilliant green color under ultraviolet light.

In this activity, you will learn about the process of moving genes from one organism to another
with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more
small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits
that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth
allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new
environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of
plasmids.

pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin.
pGLO also incorporates a special gene regulation system, which can be used to control expression of the
fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by
adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed
with pGLO DNA is accomplished by growth on ampillicin plates. Transformed cells will appear white
(wild-type phenotype) on plates not containing arabinose, and fluorescent green under UV light when
arabinose is included in the nutrient agar medium. You will be provided with the tools and a protocol for
performing genetic transformation.

Your task will be to:

1. Do the genetic transformation.

2. Determine the degree of success in your efforts to genetically alter an organism: Transformation
Efficiency.

How Can I Tell if Cells Have Been Genetically Transformed?

Recall that the goal of genetic transformation is to change an organism’s traits, also known as
their phenotype. Before any change in the phenotype of an organism can be
detected, a thorough examination of its natural (pre-transformation) phenotype must be made. Look at
the colonies of E. coli on your starter plates.

The Genes

Genetic transformation involves the insertion of some new DNA into the E. coli cells. In addition
to one large chromosome, bacteria often contain one or more small circular pieces of DNA called
plasmids. Plasmid DNA usually contains genes for more than one trait. Scientists use a process called
genetic engineering to insert genes coding for new traits into a plasmid. In this case, the pGLO plasmid
has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein,
GFP, and a gene (bla) that codes for a protein that gives the bacteria resistance to an antibiotic. The
genetically engineered plasmid can then be used to genetically transform bacteria to give them this new
trait.

Figure 1

Transformation process of plasmid DNA entering a bacteria cell

The Act of Transformation

This transformation procedure involves three main steps. These steps are intended to introduce
the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly
acquired genes.

To move the pGLO plasmid DNA through the cell membrane you will:

1. Use a transformation solution containing CaCl2 (calcium chloride).

2. Carry out a procedure referred to as heat shock.

For transformed cells to grow in the presence of ampicillin you must:

3. Provide them with nutrients and a short incubation period to begin expressing their

newly acquired genes.

Materials and Reagents for Part 1

Agar powder 0.5 g/plate and 12.5 mL of DI water/plate

Petri dishes 2 or 3 per person
125 or 250 mL Erlenmeyer Flasks 1
Hot plates 1
Magnetic Stir Bars 1
Ampicillin 125 uL for students 1-8 and 250 uL for students 9-16.
Arabinose 375 uL for students 9-16
Marking pen
Micropipettes and sterile tips
Lab 1 Procedure (Make sure to stop at the end of the lab 1 procedure)

This procedure is sequential so you will make enough agar for all 5 of your plates and add in the amp
and ara after you fill the previous plates that do not contain those two reagents.

1. Turn on your hot plate to medium-high heat to start heating it to create your agar.
2. You will be making 5 plates with your lab partner:
a. Starter plate
b. +pGLO LB/amp
c. +pGLO LB/amp/ARA
d. -pGLO LB/amp
e. -pGLO LB
3. Measure out 65.0 mL of distilled water using a graduated cylinder and pour into an Erlenmeyer flask
(make sure you clean the flask with distilled water before using – 3 rinses)
4. Weigh 2.50 g of LB nutrient Agar powder and add it to the Erlenmeyer flask with the 65.0 mL of
distilled water. Swirl to mix.
5. Add a magnetic stirrer to the flask and place the flask on the hot plate. Make sure you turn on the
magnetic stir option to the lowest setting on the hot plate.
6. As the agar is on the hot plate grab 2 petri dishes.
7. Label your plates with:
a. Your initials
b. Type of plate (Starter, LB, LB/AMP, or LB/AMP/ARA)
c. +pGLO or -pGLO
8. Heat to it boils and then reduce the heat to a low-medium setting for 10 minutes to sterilize the agar.
9. DO NOT ADD THE BOILING AGAR TO THE PETRI DISHES!
10. Place the flask on a cooling pad to let it cool to roughly 50°C. Note: Excessive heat (>60°C) will
destroy the ampicillin and the arabinose, but the nutrient agar solidifies at 27°C so one must carefully
monitor the cooling of the agar and then pour the plates from start to finish without interruption. A
general rule is if you can hold the Erlenmeyer flask in your hands for over 5 seconds and not placing it
down because it is too hot then it is good to use.
Start plate/LB Plate + LB/AMP Plate -pGLO

1. Aseptically remove the petri dish cap and place the flat top on table so the opening is facing the
ceiling. Do not touch inside the lid and base of the petri dish once opened. You need to keep them
sterile. Touch the outside only!
2. When agar is ready, aseptically pour ~12 mL (one-third to one-half full) of your agar into your starter
plate and -pGLO LB plate and put the petri dish lids back on.
3. Aseptically add 375 uL of ampicillin to the remaining LB nutrient agar in the Erlenmeyer flask and swirl
for 15 seconds.
4. Aseptically pour the ampicillin LB nutrient agar into the two plates labelled +pGLO LB/amp and -pGLO
LB/amp (one-third to one-half full) and put the petri dish lid back on.

LB/AMP Plate -pGLO + LB/AMP/ARA +pGLO

1. Aseptically remove the petri dish cap and place the flat top on table so the opening is facing the
ceiling. Do not touch inside the lid and base of the petri dish once opened. You need to keep them
sterile. Touch the outside only!
2. Aseptically add 375 uL of arabinose to the remaining LB nutrient agar in the Erlenmeyer flask and
swirl for 15 seconds.
3. Aseptically pour the LB/AMP/ARA nutrient agar into the remaining plate labelled LB/AMP/ARA +pGLO
and put the petri dish lid back on.
4. Let the plates cool to they are solidified. Should take less than 30 minutes.
5. Once solidified, stack and invert the plates so the lid is on the bottom and place them in the 4°C until
your next lab.
6. Once solidified, stack and invert the plates (put starter plate on top) so the lid is on the bottom and
place them in the 4°C until your next lab.
7. Once your plates are in the fridge clean your workstation and you are done part 1 of the
transformation lab.

END OF PART 1 OF TRANSFORMATION LAB

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Part 1.5 Rehydration of Bacteria, Streaking Starter Plates, and preparing pGLO plasmid

This part of the experiment you will perform 24 hours before our lab to grow the bacterial colonies of
E. coli HB101 on the starter plates. If we streaked the colonies in the 1 st lab then that would be two
weeks of incubation and the plates would be overgrown and the efficacy of the pGLO would be lost.

Here is the procedure I will be following to rehydrate the bacteria and streak the starter plates.

1. Rehydrate bacteria (this will be done for you)

Using a sterile pipet, rehydrate the lyophilized E. coli HB101 by adding 250 μl of LB nutrient
broth directly to the vial. Recap the vial and shake it gently to ensure all bacteria are rehydrated.
Incubate the vial at 37°C for 8–24 hr. If an incubator or waterbath is not available, the vial can be placed
into a large volume of water heated to 37°C and then left at room temperature for 16–24 hr. Store
remaining LB broth at 4°C until use.

2. Streak starter plates to produce single bacterial colonies on agar plates

Each lab team will need their own starter plate as a source of cells for transformation. This kit
contains enough material to outfit eight complete student workstations. LB plates should be streaked
for single colonies and incubated at 37°C for 24–36 hr before the transformation activity is planned.

Using the rehydrated E. coli, you prepared in the last step and eight LB agar plates (prepared in
step one), streak one starter plate for each of your student teams. The purpose of streaking is to
generate single colonies from a concentrated suspension of bacteria. A minute amount of the bacterial
suspension goes a long way. Under favorable conditions, one cell multiplies to become millions of
genetically identical cells in just 24 hr.

a. Gently shake the vial of E. coli HB101 to resuspend the bacteria. Insert a sterile inoculation
loop into the rehydrated bacterial culture. Insert the loop straight into the vial. Remove the loop and
ensure a film of bacteria is across the loop. Streak the plates as illustrated below. Streaking takes place
sequentially in four quadrants. The first streak is to just spread the cells out a little. Go back and forth
with the loop about a dozen times in the small area shown. In subsequent quadrants the cells become
more and more dilute, increasing the likelihood of producing single colonies.

b. For subsequent streaks, the goal is to use as much of the surface area of the plate as possible.
Rotate the plate approximately 45 degrees (so that the streaking motion is comfortable for your hand)
and start the second streak. Do not dip into the rehydrated bacteria a second time. Go into the previous
streak about two times and then back and forth as shown for a total of about 10 times.

c. Rotate the plate again and repeat streaking.

d. Rotate the plate for the final time and make the final streak. Repeat steps a–d with the
remaining LB plates for however many student workstations there will be. Use the same inoculation
loop for all plates. When you are finished with each plate, cover it immediately to avoid contamination.
Figure 2

Streaking Technique on Petri Plates

e. Place the plates upside down inside the incubator overnight at 37°C or at room temperature
for 2–3 days if an incubator is not available. Use for transformation within 24–36 hours as
bacteria must be actively growing to achieve high transformation efficiency. Delaying more than
36 hours will compromise transformation. DO NOT REFRIGERATE BEFORE USE.

f. E. coli forms off-white colonies that are uniformly circular with smooth edges. Avoid using
plates with contaminant colonies.

3. Prepare pGLO plasmid

Using a new sterile pipet add 250 μl of transformation solution into the vial of lyophilized pGLO
plasmid DNA. Note that the quantity of DNA is so small that the vial may appear empty. Invert the vial to
mix plasmid solution well and ensure that the DNA is dissolved and is not sticking to the cap. If possible,
store the hydrated DNA in a refrigerator. (Transformation solution is being used here because it is a
handy, sterile and nuclease-free solution. Sterile water would work just as well.)

END OF PART 1.5 OF TRANSFORMATION LAB

 Part 2 pGLO Transformation

Materials and Reagents

Transformation solution (CaCl2) 500 uL

LB Nutrient Broth 500 uL
Plates from Part 1 and Starter Plates
Crushed ice and ice container
Sterile inoculation loops 1
Bunsen burners 1
pGlO Plasmid solution (0.08 ug/ul) 10 uL
microcentrifuge tubes 2
micropipettes and autoclaves tips
water bath 42°C
Part 2 Procedure

1. Label 2 microcentrifuge tubes +pGLO and -pGLO with your initials and place them in the rack
2. Using a sterile tip, micropipette 250 uL of transformation solution into each tube.
3. Place the tubes on crushed ice immediately.
4. Using a sterile loop, pick up a single colony of bacteria from the starter plate and immerse the end of
the loop in the solution of the +pGlo tube. Twirl loop several times to disperse bacteria in the solution.
5. Sterilize the loop over the Bunsen burner and repeat step 4 for the -pGLO tube.
6. Examine the pGLO plasmid DNA solution with the UV lamp.
7. Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a loopful. There should be a
film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing
soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Optionally, pipet 10 μl of
pGLO plasmid into the +pGLO tube and mix. Close the -pGLO tube and return it to the rack on ice. Do
not add plasmid DNA to the -pGLO tube.
8. Incubate the tubes on ice for 10 minutes. Make sure the tubes are all the way down in the ice.
9. While the tubes are sitting on ice, get your agar plates ready.
10. After 10 minutes on ice grab the foam rack as a holder and transfer the tubes into the 42°C water
bath for 50 seconds. Make sure the tubes are actually in the water bath.
11. After 50 seconds place the tubes on ice for 2 minutes. Be very quick with the transfers from ice to
warm bath to ice.
12. After 2 minutes remove the tubes from the ice and use sterile tips to micropipette 250 uL of LB
broth to both tubes.
13. Allow to site at room temperate for 10 minutes.
14. Gently flick the closed tubes with your finger to mix. Using a sterile tip remove 100 uL from each
tube to the corresponding plate. i.e. 100 uL from +pGLO tube to your +pGLO plate and 100 uL from -
pGLO tube to your -pGLO plate.
15. Sterilize your loop over the Bunsen burner, allow to cool and spread the fluid evening on the plates.
Do not press hard on the plate with the loop. You do not want to damage the agar.
16. Once both plates are streaked, stack your plates, invert them and tape them together and place
them in the 37°C incubator for 24 hours.
17. Clean up your workstation.
18. You will come back to the next day (scheduled) and take photos of your plates
19. I will store the LB/AMP and LB/AMP/ARA plates for the next lab of GFP protein electrophoresis.

Analysis for lab notebook

1. Do you observe some E. coli growing on the LB plate that does not contain ampicillin or
arabinose? Explain your answer if yes or no.
2. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the
LB plate? Explain your answer.
3. What two factors must be present in the bacteria’s environment for you to see the green color?

Analysis done at home and glued/stapled to lab notebook following week

4. What is the transformation efficiency?

5. Write a discussion (minimum 150 words) on your results and how good was your transformation
efficiency. Research and provide references to validate your findings. Submit on Moodle and
you must have a similarity report below 30% in order to be accepted and add it to your lab
notebook the following week.

Calculate the transformation efficiency, which gives you an indication of how effective you were in
getting DNA molecules into bacterial cells. Transformation efficiency is a number. It represents the total
number of bacterial cells that express the green protein, divided by the amount of DNA used in the
experiment. (It tells us the total number of bacterial cells transformed by one microgram of DNA.) The
transformation efficiency is calculated using the following formula:

Therefore, before you can calculate the efficiency of your transformation, you will need two pieces of
information:

(1) The total number of green fluorescent colonies growing on your LB/amp/ara plate.

(2) The total amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate:

pGLO DNA spread in μg = Total amount of DNA used in μg x fraction of DNA used

a. The total amount of DNA we began with is equal to the product of the concentration and the total
volume used, or

(Total Amount of DNA in μg) = (concentration of DNA in μg/μl) x (volume of DNA in μl)

The values are found in the lab.

b. Determining the fraction of pGLO plasmid DNA (in the bacteria) that got spread onto the LB/amp/ara
plate: (values found in the lab)