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Transformation of E. coli using pGLO Plasmid

Amber L. Gridley

Sinclair Department of Biology

BIO 1272: Principles of Biology II

Professor John M. Erbe

September 10, 2021

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Introduction

To begin, the process of transformation has been used for a variety of

experiments in biology and other sciences. It involves changing the phenotypic

appearance of an organism by inserting a gene into the DNA. You can see

transformation in action in agriculture for pest resistance and in medicine. Gene therapy

has been a revolutionary idea that can aid in the treatment of diseases that are the

result of defective genes. Through transformation, the afflicted cells are changed into

healthy ones after beneficial genes are inserted into the DNA.

Furthermore, if there was an oil spill that needed cleaned up in an environment,

scientists can deploy bacteria to help. This process is called bioremediation and is

accomplish by giving these organisms oil-digesting genes. In our experiment, however,

we are using transformation for the process of fluorescence. For reference, when

something glows or fluoresces, it has the ability to absorb radiation and exhibit visible

light back to our eyes. Fluorescence is usually the result of the excited atoms after

absorbing the electrons from the source.

Second, in this experiment we transformed Escherichia coli or E. coli. This rod-

shaped bacterium is found in the intestine of many organisms with warm blood but can

cause infections if spread to other locations. Next, bacteria usually contain a single

large chromosome and carry a few plasmids. Plasmids are small circular DNA strands

located in the cytoplasm and are the basis of genetic engineering. In addition, these tiny

components are responsible for resistance to antibiotics since plasmids can be shared

throughout a colony.
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In order to transform our E. coli, the use of a specific plasmid called the pGLO

plasmid is critical in changing the phenotype. It was engineered by Bio-Rad and

contains all the genes necessary for bacteria to fluoresce like the source,

bioluminescent jellyfish. Overall, pGLO is responsible for coding the genes needed to

make the Green Fluorescent Protein (GFP), providing immunity to the antibiotic

ampicillin, and incorporating the correct gene regulation system. These components are

accomplished by three genes located inside the pGLO plasmid: GFP, Bla, and Ara c.

For reference, the GFP gene is responsible for manufacturing the GFP, the Bla gene

which makes beta lactamase is the cause of the antibiotic resistance, and the Ara c

gene controls the regulation system in conjunction with arabinose sugar.

Third, one universal truth in biology is that genes make proteins. DNA is

composed of thousands of genes that code for a variety of characteristics. This process

from genotype to phenotype starts when RNA polymerase comes along and reads the

strand. As a result, transcription occurs producing messenger RNA that is transported

outside the nucleus until eventually reaching ribosomes in the cytoplasm.

Next, translation is the synthesis of proteins and starts when the messenger

RNA interacts with the ribosome. Like RNA polymerase, the ribosomes are able to read

the DNA strand and then produce corresponding amino acids depending on the

sequence. Next, the amino acids are joined together by dehydration synthesis which

means the two molecules are covalently bonded, while a water molecule is lost. Finally,

the newly connected amino acids called polypeptides are arranged to form a functional

protein. They are twisted, folded, and coiled into the correct shape depending on what

their function will be in the cell.

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Materials and Methods

Starter plate of E. coli

1 LB, 2 LB/amp, and 1 LB/amp/ara agar plates

Transformation solution

LB nutrient broth

Packet of Inoculation loops

5 Pipets

Foam microcentrifuge tube holder

Foam container of crushed ice

Sharpie

2 Microcentrifuge tubes

UV flashlight

Vial of pGLO plasmids

1. Using the sharpie, label one microcentrifuge tube +pGLO and the other

-pGLO.

2. Open a clean pipet packet and add 250 µl of the transformation solution to

+pGLO and -pGLO

3. Place both tubes into the foam holder. Then, move the holder into the container

of crushed ice

4. Use a sterile inoculation loop to collect a growing colony of E. coli from your

starter plate. Insert into one of the microcentrifuges and make sure to gently twist

the loop in order to get the whole colony. Repeat this step for the second tube

with another sterile loop and place both back into the ice.
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5. Pick up another sterile inoculation loop and dip it into the pGLO plasmid DNA

stock tube. When the liquid covers the opening in the loop, mix the solution only

into the +pGLO tube and return to the container of ice.

6. Push both tubes all the way down in the foam holder in order for the liquid to

properly cool. Wait 10 minutes.

7. Using the sharpie again label one of the LB/amp plates and the LB/amp/ara plate

with a +pGLO on the lid. Next, write -pGLO on the other LB/amp plate and the LB

plate.

8. Take the rack with both microcentrifuges over to the lab counter for the heat

shock. Place the holder into the warm water, but make sure the tubes are still

sticking out of the bottom so they can make contact. Wait 50 seconds, then

quickly transfer the foam holder back into the ice container. Wait another 2

minutes.

9. Once the time is up, remove the holder from the ice and leave on the surface of

the table. Open a new sterile pipet, collect 250 µl of LB nutrient broth, and

combine into one of the microcentrifuge tubes. Repeat step using another pipet

and the other tube. Leave out in room temperature for 10 minutes.

10. Close the lids to the +pGLO and -pGLO tubes and gently flick in order to mix the

solution. Next, collect 100 µl of the appropriate transformation and control

suspensions for the agar plates. For example, +pGLO is added to the plates

labeled LB/amp and LB/amp/ara.

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11. To spread the suspensions on the surface of the agar plates evenly, use a new

loop for each container. Do not dig into the agar but move the tool quickly and

recover the plate immediately.

12. Stack the plates, tape them, and flip them upside down to reduce condensation.

Last, place them in the incubator until next class.

Results

Plate Name Colony Color Glow or No

Description Glow
Control Plate 1 Lawn of growth Creamy white No glow

(-pGLO/LB)
Control Plate 2 No growth Nothing No glow

(-pGLO/LB/amp)
Transformation Plate 4 Large colonies Only green colonies Glows

(+pGLO/LB/amp/ara)
Transformation Plate 3 Small colonies Creamy white No glow

(+pGLO/LB/amp)

Discussion
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Control plate 1 (-pGLO/LB): This version of E. coli received no pGLO plasmid but did

have the LB nutrient broth as food. As a result, there was only a lawn of growth with no

colonies. This indicates that these were the correct conditions needed for the bacteria to

grow.

Control plate 2 (-pGLO/LB/amp): Here, the organisms received no pGLO plasmid, but

the agar does contain the food and antibiotic. There was absolutely no growth on

control plate 2 (-pGLO/LB/amp) because it was never exposed to the correct genes. In

other words, this group of E. coli never received the Bla gene from the plasmid that

would have coded for a protein to protect it from the ampicillin, so they all died.

Transformation plate 3 (+pGLO/LB/amp): This was the first plate to receive the pGLO

plasmid through the heat shock method in step 8. The rapid change in temperature

increased the permeability of the cell wall and membrane to allow the plasmids in.

Although not all the cells received the pGLO genes since there were only small colonies

of creamy white growth. The few E. coli that did incorporate the correct genes were

resistant to the antibiotic because of the Bla gene, therefore, they were the ones that

replicated. They survived due to the LB nutrient broth present in their environment.

Furthermore, transformation plate 3 (+pGLO/LB/amp) did contain the Green

Fluorescent Protein and Ara c genes, but they were not turned on since the bacteria did

not glow. The Ara c protein sits on the DNA strand and acts as a repressor because it is

oriented in a way to block the promoter sequence gene. As a result, RNA polymerase

was unable to read and code the correct proteins necessary for fluoresce. The genes

were there, but without the arabinose sugar in the agar, it was impossible to

manufacture the GFP.

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Transformation Plate 4 (+pGLO/LB/amp/ara): Version 4 of the experiment received

everything needed in order to glow. First, the agar contained the LB nutrient broth

meaning it was possible for the E. coli to replicate. Second, relatively little bacteria got

the +pGLO plasmid, but were able to reproduce creating large green colonies. Next, all

the genes found in the DNA were correct and functioning. The bacteria survived despite

the presence of the antibiotic for the same reason as transformation plate 3

(+pGLO/LB/amp), the presence of Bla genes making proteins.

Finally, this batch of bacteria was able to fluoresce mostly due to the presence of

arabinose sugar in their environment. Unlike the last transformation plate, the Ara c

protein made from the Ara c gene became an activator protein. When arabinose binds

to this protein, it changes the orientation or shape which exposes the P Bad promotor.

For reference, this piece of the DNA tells RNA polymerase where to start reading in

order to activate the correct genes. So, eventually RNA polymerase makes it to the GFP

gene sequence and produces the messenger RNA during transcription. Finally,

translation occurs in the ribosomes and the chain of polypeptides is constructed into the

fully functional Green Florescent Protein.

The bacteria from transformation plate 4 (+pGLO/LB/amp/ara) will react to

ultraviolent light the same way a bioluminescent jellyfish does, by glowing. The correct

genes have been activated, producing the important proteins for the process, causing

the organisms to shine a brilliant neon green. Also, the environment had all the nutrients

required to keep this colony happy and healthy.