Katie Alpert

pGLO Transformation Lab

Introduction:
This lab uses plasmids, which are circular DNA strands that can be placed in bacterium
and are often used in genetic manipulation, to insert a gene of interest into E.Coli bacteria
cells. Plasmids are replicated when the bacteria cell duplicates along with the rest of the
cells DNA. Promoters attach to a DNA strand to begin replication from five prime to
three prime. Without a promoter, replication of the DNA will not occur and the gene of
interest of DNA will not be passed to a new generation. If antibiotics are present, bacteria
cannot reproduce. This is the reason that people are given antibiotics when they are sick
so that the bacteria that is causing the illness cannot reproduce. Antibiotic resistance is a
gene that allows bacteria to resist the antibiotic, allowing the bacteria to reproduce and
grow. Bacteria that are resistant to antibiotics are evolving as more people take antibiotics
so it is becoming increasingly difficult to cure illnesses using antibiotics. In this lab, the
resistance gene for ampicillin is added to the plasmid and placed in the bacteria. Then, the
resistance gene appears in the offspring of the bacteria. The plasmids, that had the
resistance gene for ampicillin in pGLO added to them, allow the bacteria to replicate
even in the presence of ampicillin. The GFP protein is the green fluorescent protein that
causes bacteria to glow the gene that produces this protein and makes the bacteria glow
when in the presence of arabinose. In this experiment, only one plate contained arabinose
so that was the only plate to glow when the ultraviolet light was shined on it. The plasmid
had the DNA sequence that coded for the production of the GFP protein. This is an
inducible operon because when arabinose sugar is present, the GFP protein is produced to
catalyze the sugar. In an inducible operon, the promoter attaches and begins
transformation when the inducer is present. When the sugar is broken down, the bacteria
glows. Transformation is when a plasmid is inserted with a gene of interest and then
placed back into a bacteria cell. The bacteria then replicates its DNA to reproduce, and
the offspring contains the genes that were added to the plasmid. This is a method of gene
mutation that allows scientists to breed for certain genes. In this lab, the pGLO plasmid
was inserted into two of four plates containing E. Coli. If the bacteria contains pGLO,

then it resisted ampicillin, and the ampicillin could not kill the E.Coli or inhibit its
growth. The plasmid included the DNA sequence to produce the GFP protein in addition
to the ampicillin resistance gene.
E. Coli Growth in Plates that Either Contain or Lack the pGLO Plasmid:
+pGLO

+pGLO

-pGLO

-pGLO

LB/amp

LB/amp/ara

LB/ amp

LB

https://johnflory.wordpr
-Large amounts of

-large colonies and

ess.com/
-No growth

growth and

a great amount of

-does not glow, same

-most growth

massive colonies

growth

color under UV light

-growth covering

-tan color

-tan but glows

entirety of plate

-does not glow,

green/ yellow

-tan color

same color under

-Lawns of growth

-does not glow,

UV light

same color under

-There was not as

UV light

much growth as the

-This is the control

control group

group so this

because not all of

depicts how E.

the bacteria

Coli normally
grows.

Conclusion:
The transformation was successful because both of the plates that had bacteria that
contained the plasmid grew even though they were in an environment with ampicillin.
Additionally, the plate contained that arabinose had glowing E. Coli meaning that the
transformation was successful because the gene of interest, the GFP protein and the
ampicillin resistance gene, is present in the E. Coli. The first plate, -pGLO LB, is a
control plate to tell if the E. Coli can grow without outside materials, which it did. The
second dish, -pGLO LB/amp, shows that ampicillin kills E. Coli if there is no resistance
gene present. There was no growth in this plate, meaning ampicillin kills E.Coli. The next
dish, +pGLO LB/amp, displays that the resistance gene in the pGLO counteracts the
ampicillin, allowing the E. Coli to grow. The last dish, +pGLO LB/amp/ ara, is used to
show that when arabinose sugar is present, the GFP protein that is coded for in the
plasmid will be produced to break down the sugar, thus making the E. Coli on this plate
glow. Next time, I would ensure that the ampicillin in the LB/amp plate with the pGLO
was living so that it accurately depicts what should occur when a resistance gene is not
present, no growth. Additionally, I would test the affects of a positive pGLO in just
arabinose to see what happens to the E.Coli if no ampicillin is present. It is beneficial for
organisms to be able to turn genes on and off because they will not waste energy
producing proteins that they do not need. If organisms use operons to regulate the
transcription of mRNA, they will only produce proteins when a protein is not available in
the environment or an enzyme is needed to break down materials that are in the
environment. Transforming bacteria can be used to genetically modify crops so that they

are resistant to pests or drought. Additionally, the DNA of bacteria can be changed to
consume oil to clean up oil spills. Lastly, genetic modification can cause animals such as
rabbits, to glow, so scientists can use this knowledge to modify the DNA of organisms
and breed for specific traits.