Genetic Transformation of E. Coli with the Green Fluorescent Protein
Kaelyn Schneider
Section 3
Introduction
The purpose of this experiment was to perform genetic transformation on E. Coli bacteria with a
gene that codes for Green Fluorescent Protein. All bacteria typically contain small, circular
pieces of DNA called plasmid, which contain genes. It is important to know that a gene is a piece
of DNA that provides instructions on how to make a protein, which gives an organism a trait to
help it survive. The plasmid used in this experiment is called pGLO and it codes for the Green
Fluorescent Protein gene, which comes from bioluminescent jellyfish and gives them a trait to
allow them to glow in the dark. The pGLO also codes for resistance to the antibiotic ampicillin,
therefore producing E. coli growth on plates treated with ampicillin. Genetic transformation
occurs when a gene is inserted into an organism, causing one of its traits to change. The GFP
gene activates in transformed cells by adding the sugar arabinose to a cell’s nutrient medium.
Because GFP is resistant to ampicillin and causes fluorescence, transformation will be successful
if the affected bacteria grow on a plate containing ampicillin and glow under UV light if
arabinose is present. It was hypothesized that both the -pGlo LB and +pGlo LB plates would
show bacterial growth along with the +pGlo LB/ampicillin plate and that the +pGlo
LB/ampicillin/arabinose plate would show growth and glow, proving genetic transformation
occurred.
Materials and Methods
Materials:
Material
E. Coli Starter Plate
Poured Agar Plates (2LB, 2LB/Amp,
2LB/Amp/Arabinose)
Transformation Solution CaCl2
LB Nutrient Broth
Inoculation Loops
Pipets
Plastic Microcentrifuge Tube Holder
Ice Bucket With Crushed Ice
Marking Pen
Microcentrifuge Tubes
Rehydrated pGlo Plasmid
42 C Water Bath With Thermometer
UV Light
37 C Incubator
2-20 l adjustable micropipettes
2-20 l micropipette tips
Quantity
1
6
1
1
10
5
1
1
1
2
1 vial
1
1
1
2
1 box
Methods:
The procedure for this experiment was obtained from the “Lab Ex. #6: pGlo Transformation”
packet given in class, specifically on pages 5-9.
Creating the Suspensions:
Two test tubes were created; both containing 250 l of the transformation solution CaCl2 and
roughly 2-4 colonies of the E. Coli bacteria. One tube was labeled +pGLO, where 5 l of the
pGLO plasmid was added and the other tube was labeled -pGLO where no plasmid was added.
Heat Shock:
A heat shock was performed on both tubes where they were put on ice for 10 minutes, then
placed into a 42 C water bath for exactly 50 seconds. Immediately after this, the test tubes were
put back on the ice for 2 minutes. This process is important because increasing the temperature
creates pores in the plasma membrane of the E. Coli and allows DNA to enter the bacterial cell.
Next, 250 l of LB nutrient broth was added to each test tube and they were incubated at room
temperature for 10 minutes. The tubes were then flicked gently to mix and resuspend the
bacteria. The LB nutrient broth is necessary because it is used to culture the bacteria and aid in
its growth.
Creating the Plates
Next, a set of three control plates was created; one with only LB agar, one with LB agar and
ampicillin, and finally one with LB agar, ampicillin, and arabinose sugar. No pGLo was added to
any of the plates. The second set of plates was the transformation plates. They were exactly the
same as the control plates except 100 l of the +pGLO was spread evenly on each plate using a
sterile loop. The plates were covered, stacked, and placed upside down in a 37 incubator. A
week later, each plate was observed under both normal room light and under a handheld
ultraviolet light.
Results:
Table 1: Control Plates
Bacterial Growth
Bacteria Color
Estimated Number
of Colonies
-pGlo LB
-pGlo LB/ampicillin
++ (moderate
growth)
White/Yellow
7-8 colonies
-(no growth)
-pGlo
LB/ampicillin/arabinose
-(no growth)
n/a
0 colonies
n/a
0 colonies
+pGlo LB/ampicillin
+pGlo
LB/ampicillin/arabinose
+++ (profuse growth)
Yellow, fluorescent
green under UV light
Too many to count
Table 2: Transformation Plates
+pGlo LB
Bacterial Growth
Bacteria Color
+ (little growth)
White/Yellow
n/a
n/a
Estimated Number
2 colonies
n/a
of Colonies
*Note: No data was able to be recorded for the +pGlo LB/Ampicillin plate because there was no
plate created due to a shortage of materials.
Interpretation: Each of the six plates was observed under normal light and ultraviolet light. The
three key observations made were the amount of bacterial growth, color of bacteria, and the
estimated number of colonies. Table 1 showed data for the control plates. The -pGlo LB plate
showed moderate bacterial growth and around seven to eight colonies were observed. The
colonies were yellow/white. Both the -pGlo LB/ampicillin and -pGlo LB/ampicillin/arabinose
displayed no growth, therefore zero colonies were counted, and no bacteria was present. Table 2
showed data for the transformation plates. The +pGlo LB plate showed little bacterial growth
with only two estimated colonies. They were also yellow in color. No data for the +pGlo
LB/ampicillin was recorded. The +pGlo LB/ampicillin/arabinose plate was significant because it
produced the most bacterial growth. It was recorded that this plate had profuse E. Coli growth
and that there were too many colonies to count accurately; they were also yellow in color.
Discussion:
It was hypothesized that bacterial growth would occur on the -pGlo LB, +pGlo LB, and
+pGlo LB/ampicillin plates, whereas the +pGlo LB/ampicillin/arabinose plate would display
bacterial growth and glow under the UV light, proving that genetic transformation had occurred.
Based on the data recorded and observed, this hypothesis was mostly supported. All of the
expected results remained accurate, except that there was no growth on the +pGlo LB/ampicillin
plate, simply because there was no plate to observe. Both the -pGlo LB and +pGlo LB plates
showed bacterial growth. Also, the +pGlo LB/ampicillin/arabinose plate showed expected
results, as there was bacterial growth and the colonies glowed.
The method used was to create a group of control plates and a group of transformation
plates so that a comparison could be made. The control plates were treated with a mixture of
CaCl2, E. Coli colonies, and the LB nutrient. The transformation plates were treated with the
same exact mixture as the control plates, just with the pGlo plasmid added. Each group consisted
of the same three plates; one treated with LB, one with LB and ampicillin, and one with LB,
ampicillin, and arabinose sugar. The reason for this was to prove that the pGlo plasmid was
required for the bacteria to grow on the plates with ampicillin. Also, the -pGlo plate proves that
the starter cultures not treated with the pGlo are unable to grow on the LB/ampicillin plate. This
method was effective because it allowed for comparison and proved that the genetic
transformation did take place in the experiment. It was limited in that error could have occurred
in many ways such as contamination, or if the bacteria didn’t take up the plasmid. Data was
collected strictly based on observation.
Both the -pGlo LB and +pGlo LB plates served as standards for both sets of plates.
Because they had LB, the bacteria were able to grow normally on these plates. It did not matter
whether they had the plasmid or nor because LB cultures the growth of normal bacteria. The pGlo LB/ampicillin plate showed no growth because the bacteria did not have the plasmid that
made it resistant; therefore, the ampicillin killed the E. Coli. There was no plate for the +pGlo
LB/ampicillin plate, but it can be assumed that bacterial growth would occur because the
presence of the plasmid would make the bacteria resistant to the ampicillin. The -pGlo
LB/ampicillin/arabinose plateshowed no growth because the E. Coli had no plasmid and was not
resistant to the ampicillin. The arabinose on this plate had no effect because the bacteria couldn’t
survive. Lastly, the +pGlo LB/ampicillin/arabinose plate showed the most growth and glowed
because the plasmid resisted the ampicillin and the presence of the arabinose activated the Green
Fluorescent Protein. This was the only plate where genetic transformation took place.
One prominent error in this experiment was a shortage of materials. There was no +pGlo
LB/ampicillin plate so no data could be collected, and no observations could be made. Also, this
plate could not be used as a comparison to any of the other plates. Other errors could have taken
place such as inexact measurements when pipetting or inaccurate timing during the heat shock.
Overall, no extreme known errors occurred that altered the results of the experiment. For the
most part, the results obtained were expected.
In conclusion, genetic transformation of the E. Coli for the Green Fluorescent Protein did
occur in this experiment. Bacteria was able to grow in normal conditions with and without the
pGlo plasmid added. Bacteria with the pGlo added was able to resist the presence of ampicillin
and continue to grow. Bacteria with the pGlo added on the plates with ampicillin and arabinose,
grew and glowed proving that the addition of the plasmid gave the bacteria a new trait, that being
the ability to fluoresce. This experiment can be useful in further testing of organismal traits and
determining if organisms can turn genes on or off in response to certain conditions.
Sources:
Bacterial Transformation. (n.d.). Retrieved from https://www.addgene.org/protocols/bacterialtransformation/.
Lab Instructions. (n.d.).