Title: The Effect of pGLO on the Gene Expression of GFP and Antibiotic Resistance
MIST ID’s: 1745-11024 (FK)
Abstract:
Biotechnology is the manipulation of an organism's genes to create beneficial
products. Genetic engineering is a branch of biotechnology that describes gene cloning through
manipulation of specific genes for practical uses. The problem addressed in this experiment was
“What will cause GFP (gene) to be expressed in E.coli bacteria?” It was hypothesized that if E.
coli bacteria containing the pGLO plasmid are grown in a nutrient agar solution containing sugar
arabinose and ampicillin (antibiotic), then the bacteria will glow under ultraviolet light,
indicating the presence and expression of GFP and acts expression. The plasmid pGLO also
encodes a gene responsible for bacterial resistance to the ampicillin. The experiment involved
the use of four plates. The first one had nutrient agar, ampicillin, and E. coli that contained the
pGLO. The second plate had nutrient agar, ampicillin, arabinose, and E. coli that contained
pGLO. The third plate had nutrient agar, ampicillin, and E. coli that did not contain pGLO. The
fourth plate had nutrient agar and E. coli that did not contain pGLO. Results supported this
hypothesis since the E. coli bacteria had pGLO inserted inside of them and were grown in the
plate containing sugar arabinose and ampicillin, survived and glowed under the UV rays,
indicating the presence of the gene GFP product.
Introduction:
Genes are fragments of DNA that contain instructions for making proteins. The proteins
give an organism a certain trait. An organism’s trait can be altered through the use of genetic
engineering. Bacterial plasmids are tiny, circular DNA molecules that can carry any gene. They
replicate independently from a much bigger bacterial chromosome and are passed from one
bacteria generation to the next. Due to such characteristics, plasmids are key material for gene
manipulation and gene cloning which is the creation of numerous identical copies of a genecarrying piece of DNA (Campbell, et al, 2009).
Biotechnology is the manipulation of organisms or their parts to create beneficial
products. Scientists and researchers can insert certain favorable genes into plasmids, forming
recombinant DNA (how? explain). They can then harvest the desired product of the gene if the
recombinant bacteria replicated into a clone. The tools used to create recombinant DNA include
restriction enzymes and DNA ligase. Restriction enzymes cut DNA at certain sequences called
restriction sites to form restriction fragments while DNA ligase attaches the fragments together
(Campbell, et al, 2009).
Genetic engineering is a branch of biotechnology that describes gene cloning through
manipulation of specific genes for practical uses. To be able to manipulate genes in a laboratory,
biologists usually use bacterial plasmids since they replicate separately from the much bigger
bacterial chromosome and are passed from one bacteria generation to the next (Campbell, et al,
2009). An example of a practical use of genetic engineering is the creation of live attenuated
vaccines to work against some viral agents such as human pathogens which include influenza,
measles, mumps, rabies,respiratory syncytial, Ebola virus, and hantaviruses (Palese, et al, 1996).
This whole para basically repeats itself from the previous, must be combined.
Through the use of recombinant DNA technology and genetic engineering, certain genes
can be inserted into bacteria for replication. An example of such a gene is a pGLO which codes
for Green Fluorescent Protein (GFP). GFP originates from the bioluminescent jellyfish
Aequorea victoria. The pGLO can be inserted inside bacteria to be passed onto other bacterial
generations and expressed. When GFP is expressed in bacteria, the bacteria glow a green color
under ultraviolet light. pGLO also encodes a gene for resistance to the antibiotic ampicillin. The
gene for GFP can be switched on in transformed cells through the addition of sugar arabinose to
a cells nutrient medium. Arabinose is a promoter that causes expression of GFP.
The problem being addressed was what will cause GFP to be expressed in E.coli
bacteria? It was hypothesized that if E.coli bacteria containing the pGLO plasmid are grown in
a nutrient agar solution containing sugar arabinose and ampicillin, then bacteria will glow in the
dark, indicating a the presence of GFP.
Materials and Methods:
One micro test tube was labeled +pGLO while another micro test tube was labeled –
pGLO. The two tubes were then placed into a foam tube rack. The tubes were then opened and a
sterile Eppendorf pipet was used to transfer 250 microliters of transformation solution, CaCl2,
into each tube. The foam rack and tubes were then transferred to a container filled with ice. A
starter plate containing E. coli was taken and a sterile loop was then used to pick up a single
colony of bacteria from it. The single colony of bacteria on the sterile loop was then immersed
into the transformation solution at the bottom of the +pGLO tube. The loop was shaken inside
the tube until the entire colony had dispersed in the transformation solution with no floating
chunks. The tube was then closed and placed back inside the tube rack on ice. Using a new
sterile loop, another single colony of bacteria was picked up from the started plate and immersed
into the transformation solution at the bottom of the -pGLO tube. The loop was shaken inside
the tube until the entire colony had dispersed in the transformation solution with no floating
chunks. The tube was then closed and placed back inside the tube rack on ice.
Next, ten microliters of the pGLO plasmid was removed and added only to the micro test
tube labeled +PGLO. The ten microliters were mixed into the cell suspension of the +pGLO
tube. The tube was then closed and placed back onto the ice rack. No plasmid was added to the
tube labeled –pGLO. The tubes were pressed all the way to the bottom of the rack so that they
could touch to ice. Both the tubes were then incubated on ice for ten minutes. While the tubes
were being incubated, four LB nutrient agar plates were labeled on the bottom of the petri
dish. The first one was labeled LB/amp +pGLO. The second plate was labeled LB/amp/ara
+pGLO. The third plate was labeled LB/amp –pGLO. The fourth plate was labeled LB –
pGLO. The LB/amp +pGLO and LB/amp/ara +pGLO plates served as the experimental plates
since they contained the pGLO. The LB/amp –pGLO and LB –pGLO plates served as the
controls since they did not contain any pGLO.
After the tubes had been incubated on ice for ten minutes, both tubes were transferred
into a water bath that was set at 42oC for exactly fifty seconds. It was made sure that both tubes
were pushed all the way down into the rack to make contact with the hot water. After the fifty
seconds, both the tube were picked up and placed back onto the ice rapidly. The tubes were then
incubated on ice for another two minutes. The rack containing the tubes were then removed
from the ice and placed on a bench top. One of the tubes was opened and a new sterile pipet was
used to transfer 250 microliters of LB nutrient broth into it. The tube was then closed as this
process was then repeated with the other tube. Both tubes were then incubated for ten minutes at
room temperature.
After the ten minutes were over, both closed tubes were taped with a finger to mix their
contents. Then, a sterile pipet was used to pipet out 100 microliters of the transformation and
control suspensions onto the appropriate nutrient agar plates. Then, each of the LB nutrient agar
dishes was opened and six glass beads were placed into them. The beads were then pushed back
and forth across each of the nutrient agar dishes to spread the suspensions evenly around the
surfaces. After about two minutes, the plates were quickly inverted over a beaker to remove the
glass beads. All four plates were then stacked up and taped together. The plates were all stacked
upside down into a 37oC incubator for the next day. The next day, the agar plates were taken out
of the incubator and observed under UV lights. Observations and data were taken down and
recorded.
Results:
Figure 1: Transformation Plate : +pGLO LB/amp
Figure 2: Transformation Plates : +pGLO LB/amp/ara
Figure 3: Control Plate: -pGLO LB/amp
Figure 4: Control Plate: -pGLO LB
Table 1: Observations of the Bacterial Transformation using the pGLO plasmid
Plate Title
+pGLO LB/amp
+pGLO LB/amp/ara
Observations

34 colonies

Did not glow under UV light

27 colonies

Colonies grew under UV light
-pGLO LB/amp

No growth
-pGLO LB

Lawn growth everywhere

Did not glow under UV light
ADD GRAPH OF COLONY GROWTH, LAWN GROWTH = ?
The nutrient plates were observed under a UV light for gene expression of pGLO. It was
found that the plate with pGLO, LB, and ampicillin had growth of bacteria but the bacteria did
not glow under the ultraviolet light, indicated that GFP was not present (Figure 1) (Table 1). It
was also found that the plate with pGLO, LB, and ampicillin, and ara had growth of bacteria and
the bacteria did glow under the ultraviolet light, indicating that GFP was present (Figure 2)
(Table 1). The plate with LB, ampicillin, and no pGLO had no growth of bacteria, indicating that
conditions were not suitable for the growth of bacteria colonies (Figure 3) (Table 1). The plate
with LB and no pGLO had growth all over the nutrient agar dish, but none of the bacteria glowed
under the ultraviolet light, indicating that GFP was not present (Figure 4) (Table 1).
Discussion:
There were two transformation plates, +pGLO LB/amp and +pGLO LB/amp/ara. The
plate with pGLO, LB, and ampicillin had growth of bacteria but the bacteria did not express the
gene for GFP (Figure 1) (Table 1). Although ampicillin is used to kill bacteria, the bacteria on
this plate survived since they pGLO was inserted inside them. pGLO encodes a gene for
resistance to the antibiotic ampicillin, allowing the bacteria to survive in such a fatal
environment (HOW?). However, even though pGLO was present inside the bacteria and also
encodes for the gene for GFP, the bacteria did not express the gene for GFP since arabinose was
not present. Araribonose serves as a promoter that causes expression of GFP (EXPLAIN). The
plate with pGLO, LB, and ampicillin, and ara had growth of bacteria that did express the gene
for GFP (Figure 2) (Table 1). Although these bacteria were also placed inside a plate containing
ampicillin, they survived due to the insertion of the pGLO plasmid inside them. They also
expressed the gene for GFP since they were grown in a plate that contained arabinose.
There were also two plates that served as controls (OF WHAT?), -pGLO LB/amp and pGLO LB. The plate with LB, ampicillin, and no pGLO had no growth of bacteria (Figure 3)
(Table 1). This occurred since as mentioned before, ampicillin kills bacteria and the bacteria had
no pGLO plasmid for resistance. The plate with LB and no pGLO had bacterial growth all over
the nutrient agar dish, but none of the bacteria expressed the gene for GFP (Figure 4) (Table 1).
This occurred since these bacteria contained no plasmid. This meant that they had no factor to
abolish their survival. These bacteria also did not express the gene for GFP since they did not
have the pGLO plasmid.
The reason for the use of ampicillin was to check if pGLO had been successfully inserted inside
the bacteria, allowing them to survive in a fatal environment. Arabinose was a promoter and was
used to cause the expression of GFP.
The problem being addressed was what will cause GFP to be expressed in E.coli
bacteria? It was hypothesized that if bacteria containing the pGLO plasmid are grown in a
nutrient agar solution containing sugar arabinose and ampicillin, then bacteria will glow in the
dark, indicating a the presence of GFP. Results supported this hypothesis since the E.coli
bacteria that contained pGLO and were grown in the plate containing sugar arabinose and
ampicillin, survived and glowed under the ultraviolet rays, indicating the presence of the gene
GFP. It can also be concluded that arabinose causes the expression of the GFP protein in bacteria
with pGLO inserted inside them. The next step question that was asked after this experiment had
been conducted was can the plasmid pGLO be inserted in other types of bacteria, other than
E.coli?
To improve this experiment, other controls should have been used such as -pGLO
LB/ara, +pGLO LB/ara, –pGLO LB/amp/ara, and +pGLO LB. The control of -pGLO LB/ara
would have shown that arabinose cannot cause the expression of GFP if the plasmid pGLO is not
inserted. The control of +pGLO LB/ara would have shown lawn growth of bacteria expressing
the gene for GFP since there is no ampicillin present to kill any bacteria. The control of –pGLO
LB/amp/ara would have shown that pGLO causes ampicillin resistance and that arabinose only
depends on pGLO. The control of +pGLO LB would have shown that arabinose is needed to
cause expression of the gene for GFP.
A source of error that may have occurred was that the beads may have not spread the
bacteria over the plate evenly. Another source of error that may have occurred was that
contaminants may have accidently entered the agar dishes when they were opened.
References:
1. Campbell, N. A., Reece, J. B., Taylor, M. R., Simon, E. J., & Dickey, J. L. (2009).
Biology: Concepts and Connections (6th ed., pp. 232-251). San Francisco: Benjamin
Cummings.
2. Palese P., et al., (1996). Negative-strand RNA viruses: Genetic engineering and
applications. Proc. Natl. Acad. Sci. USA 93() 11354-11358
3. O'Donnell W. M., (2003). Inducing Ampicillin Resistance in Escherichia coli.
Transactions of the Kansas Academy of Science 106 (1/2) 99-104