E.

Coli pGLO Transformation

Pravallika Andhavarapu

Advanced Placement Biology

Period 3

Glady Ruiz

April 2nd, 2024

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Abstract

The purpose of this experiment is to understand the process by which Escherichia coli

transforms with the uptake of the pGLO plasmid from Aequorea victoria and to explore the

conditions that trigger the bacteria to glow in the presence of ultraviolet light. Out of the four

lanes: lane 1 (-pGLO/LB), lane 2 (-pGLO/LB/amp), lane 3 (+pGLO/LB/amp), and lane 4

(+pGLO/LB/amp/lac), lanes 1, 3, and 4 are expected to grow and lane 4 is expected to glow

under blue light. However, our results yielded one glowing colony in lane 4 and no growth in

lanes 1-3. Despite the unexpected lack of growth in lanes 1 and 3, the successful growth and

luminescence under UV light of our single colony in lane 4 confirmed our hypothesis. The

surprising lack of growth in the remaining lanes suggests further investigation into the

methodology and conditions of E. coli transformation and growth.

Introduction

Plasmids are small, supplementary, and circular double strands of DNA that contain

operons. Operons are short structures in prokaryotes that contain regulatory processes of the

gene. The plasmid that was inserted into Escherichia coli, a rod-shaped bacteria that is usually

found in the lower intestine of mammals, from Aequorea victoria contains several genes: Green

Fluorescent Protein GFP, Beta Lactamase bla, and araC Regulatory Protein. GFP is the protein

in the jellyfish that exhibits an intensified green fluorescence when exposed to UV light, bla

provides Ampicillin resistance, and araC regulates the transcription of GFP by activating it in

the presence of sugar (lactose) (Bacterial Transformation Lab, n.d.).

Within the realm of bacterial transformation, the first of its type was performed by British

bacteriologist Frederick Griffith in 1928. This laid the foundation for the understanding of how
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bacteria can change their form and function via the process of uptaking genetic material from

their environment (Griffith’s Experiment, n.d.). This unique ability evolved in bacteria overtime

because it provided a competitive advantage; it’s like an almost-permanent “power-up”–as they

generally can’t eject the plasmid once it has been absorbed–which allows the bacteria to take on

new abilities. The implications for this in the biomedical field are numerous, such as with the

transformation of E. coli by inserting the plasmid pBR322 to produce insulin (Riggs, 2021).

Despite the countless innovations in this field, there is much to learn about the conditions

from which transformation can occur. By recreating bioluminescent E. coli, this study aims to

delve into the conditions that favor bacterial transformation in alteration of E. coli with the

pGLO plasmid from Aequorea victoria and the conditions required for its fluorescence.

The null hypothesis posits that the uptake of the pGLO plasmid, the procedure, and

condition by which the transformation is performed will not affect the bioluminescence of E.

coli. In contrast, the alternative hypothesis suggests that the conditions and procedure–in addition

to the uptake of the pGLO plasmid by E. coli–will influence its luminescent properties.

Materials

The following materials (MiniOne, n.d.) were used over the course of a single day when

the experiment was performed during class:

● 1 x gloTray® with LB agar culture media

● 1 pack of sterile wooden spreaders

● 1 x 0.65 mL microcentrifuge tube with aliquoted overnight culture (approximately

400 µL)

● 1 x yellow PCR tube with 10 μL eGFP plasmid DNA

● 1 x blue PCR tube with 10 μL dH2O

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● 1 x clear 0.65 mL tube with 100 μL CaCl2

● 1 x green 0.65 mL tube with 50 μL Lactose

● 1 x 20-200 μL adjustable volume micropipette

● 1 x 2-20 μL adjustable volume micropipette

● 1 x rack 2-200 μL pipette tips

● Racks for PCR tubes and 0.65 mL microcentrifuge tubes

● Fine point permanent marker

● Waste container

● Visual Protocol Worksheet to Annotate

● Apron

● Eye protection

Common workstation shared by class:

● Starter culture in LB broth

● MiniOne® PCR Systems

● Tablets with MiniOne® PCR App

● Benchtop microcentrifuges

● Incubator set to 30°C

Methodology

Before the day of the lab: PCR protocol was prepared, tested, and linked (for each

protocol, final incubation temperature was 4 degrees Celsius):

● Protocol 1 (600 seconds incubation at 4 degrees Celsius)

● Protocol 2 (45 seconds heat shock at 42 degrees Celsius)

● Protocol 3 (120 seconds incubation at 4 degrees Celsius)

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One the day of the lab:

● Removed the lid and sealing film from the gloTray®. Discarded the film but kept

the lid.

● Labeled lanes as followed: lane 1 (-pGLO/LB), lane 2 (-pGLO/LB/amp), lane

3(+pGLO/LB/amp), and lane 4 (+pGLO/LB/amp/lac).

● Using 2-20 µL pipette, 25 µL of lactose was added from the green PCR tube only

into lane 4. Tilted the gloTray® back and forth to spread the lactose evenly.

Closed the lid to prevent contamination.

● Labeled yellow PCR tube with plasmid +pGLO and the blue PCR tube H20.

● Centrifuged cell culture tube for 2 minutes in benchtop microcentrifuge

(minimum 6,000 RPM). Made sure the centrifuge was balanced with tubes from

other groups.

● Removed supernatant with a 20-200 µL pipette and discarded in the sink.

● Added 60 µL of CaCl2 into the PCR tube with the pellet. Pipetted up and down a

few times to resuspend the pellet.

● Added 30 µL of the cell suspension to the +pGLO tube. Took a few attempts to

measure out and fill the tube, so resuspension was done poorly.

● Changed pipette tip and added 30 µL of the cell suspension to the H20 tube. Took

a few attempts to measure out and fill the tube, so resuspension was done poorly.

● Turned on the PCR machine and placed tubes inside.

● When protocol was finished, tubes were quickly removed from the machine and

distributed into respective lanes (separate 2-20 µL pipettes were used): lanes 1
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and 2 received the H20 tube, while lanes 3 and 4 received the +pGLO tube (15 µL

into each lane).

● Spreaded liquid in each lane with separate wooden spreaders.

● Trays were labeled, covered and incubated overnight at 33°C +/- 2°C with the

bottom of the tray facing upwards.

Viewing bacteria after incubation:

On viewing day, the gloTray® lid was removed and the tray was placed into the Wilson

Fluorescence Reader with the surface of the agar facing upwards.

All materials and procedural steps were provided in class by the MiniOne Bacterial

Transformation Lab Protocol (MiniOne, n.d.).

Results

Table 1a

Expected Bacteria Transformation

Lane Lane 1: Lane 2: Lane 3: Lane 4:

-pGLO/LB -pGLO/LB/amp +pGLO/LB/amp +pGLO/LB/amp/lac

Relative Growth Normal No growth (-) Little Growth Little Growth (+)
of E. coli (-, +, ++, Growth (++) (+)
or +++)

Bioluminescence No No No Yes
under UV Light
(Yes or No)
Note. Table 1a is based on the understanding that lane 1 (-pGLO/LB) behaves as a positive

control, which is expected to have normal growth. Lane 2 (-pGLO/LB/amp) behaves as a

negative control to negate the possibility of the ampicillin behaving non-selectively. Lane 3

(+pGLO/LB/amp) shows the vital function of the araC gene in bioluminescence; without the

presence of sugar (lactose), the GFP gene will not start transcription and will not created GFP,
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which is responsible for the glow. Lane 4 (+pGLO/LB/amp/lac) has the sugar and exhibits

bioluminescence, unlike Lane 3. There are fewer colonies of E. coli in lanes with ampicillin

because this antibiotic selects for transformed bacteria, which have the bla gene for resistance.

Table 1b

Actual Bacteria Transformation

Lane Lane 1: Lane 2: Lane 3: Lane 4:

-pGLO/LB -pGLO/LB/amp +pGLO/LB/amp +pGLO/LB/amp/lac

Relative Growth No growth (-) No growth (-) No growth (-) Little growth: one
of E. coli (-, +, ++, colony (+)
or +++)

Bioluminescence No No No Yes
under UV Light
(Yes or No)
Note. Table 1 depicts the expected results in comparison to the actual results of the

transformation with relative growth measured by no growth (-), little growth (+), normal growth

(++), and lawns (+++). The table also depicts the property of bioluminescence under UV light

when placed in the Wilson Fluorescence Reader of each of the lanes. The properties of the lanes:

-pGLO/LB (lane 1) , -pGLO/LB/amp (lane 2), +pGLO/LB/amp (lane 3), and

+pGLO/LB/amp/lac (lane 4).

Between Table 1a and Table 1b, there are notable differences between the the growths of

lanes 1, and 3. However, the expected results (Table 1a) and actual results (Table 1b) show no

difference in regards to the bioluminescence.

Figure 1a

Bacterial Growth Under Daylight

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Figure 1b

Bacterial Growth Under UV Light in Wilson Fluorescence Reader

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Note. Lanes (1-4) increase from left to right and are oriented to match between Figure 1a, Figure

1b, and Figure 2. Lanes consist of: lane 1 (-pGLO/LB), lane 2 (-pGLO/LB/amp), lane 3

(+pGLO/LB/amp), and lane 4 (+pGLO/LB/amp/lac).

Figure 2

Peer Bacterial Growth Under UV Light in Wilson Fluorescence Reader

Note. Illustrates bacterial growth data obtained from Group 5’s experiment, which yielded more

successful results.

Between Figure 1 and Figure 2, it is apparent that there were some discrepancies in

growth, considering the differences in growth of lanes 3 and 4 in Figure 2 in comparison to

Figure 1. However, the growth in lane 1 remains at a consistent zero between the figures. This is

peculiar in comparison to the expected results in Table 1a, which predicts normal growth in lane

1.

Table 3
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Class Results for Bacterial Growth by Group for Lanes 3 and 4

Group Number Lane 3 Lane 4

3 53 89

4 0 1

5 20 10

7 1 1
Note. Amount of bacterial growth is measured in colonies. Group 3 had many colonies, so the

numbers listed there are relative. It was concluded in class that Group 7 was centrifuged for more

than 10 minutes, which yielded these results. Group 4 was the group that performed the

experiment in this lab report, hence the red coloring.

Table 3 shows varied success rates in terms of bacterial growth, ranging from 0 to 89

colonies. The discrepancies and notable differences stand out, with Group 5 exhibiting average

growth, Groups 7 and 4 being extremely low, and Group 3 being extremely high (outlier).

Discussion

Primarily, it stands that there is little to no growth in lanes 1,3, and 4, even though there

should be growths ranging from normal to little. The results amongst our peers are various; there

were those with similar growth, somewhat higher, and exponentially higher bacterial growth.

With these results, it is fair to suggest that the null hypothesis is false; while not much of

E. coli bacteria grew, the single colony in lane 4 did in fact glow, implying that there is a

connection between the transformation of the bacteria and bioluminescence (alternative

hypothesis approved). Like the results displayed, the only growth out of the whole tray was in

lane 4 (+pGLO/LB/amp/lac), contrary to what we expected (see Table 1). Group 4 firstly

considered the fact that resuspension of the plasmid was a source of error; however, when

dispensing the bacteria into and out their respective vials, we had pipetted and released the
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solution a few times because of our inexperience in pipetting. So while our group did not

intentionally resuspend during the procedure, our acts of uncertainty surely behaved the same as

if we were to intentionally resuspend. Due to the fact that the whole class received E. coli from

the same tray, which was incubated overnight at 32 degrees Celsius, it is safe to eliminate the

bacteria itself as being faulty, as some of our peers had better success in growthing their bacteria.

In addition to this, there may or may not have been an issue with the plasmid itself, since our

only growth had the plasmid. The lack of growth in lane 1 also supports the idea that the plasmid

was not a source of error, as it did not contain one. It is possible that something procedurally

could have affected the viability of the bacteria in lane 1, given that there was no selection

pressure. The next logical conclusion is that Luria Broth could have been contaminated or was

ineffective in nurturing the bacteria, especially because if there were to be any sort of growth, the

easier lane to thrive in would be lane 1, which serves as our positive control. The LB was in

every lane, so an issue here would affect the growth of bacteria in the whole tray.

In correlation to Griffith’s experiment and the complexity of this one, it is important to

note that bacterial transformation typically occurs between bacteria (horizontal gene transfer),

which can explain why the uptake of plasmids from other species is an arduous procedure.

Bacterial transformation has evolved to give bacteria a competitive advantage, in which it

shouldn’t have too many conditions to occur. Nature is nothing like a lab setting; it is

uncontrollable, unpredictable, and has numerous variables to consider. That being said, it begs

the question of how complicated this procedure is in order to be successful. There are other

factors at play, mainly the state of the bacteria and the type of plasmid being used. Since

experiments akin to this one typically aim to transform bacteria into something they are not

evolved to do, procedures get complicated and have a variety of possible errors at every turn that
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could result in unsuccessful transformation. The procedure of bacterial transformation would be

considerably less complicated if we were transforming them using native plasmids (from other

bacteria completely rather than having components of other species), as they would be more

compatible, though doing so may defeat the purpose of doing the transformation at all. This is

because such transformations are done to achieve a particular purpose beyond seeing whether it

can be done or not; its primary purpose is to produce results in the form of a trait or a product

(such as a protein). If selected genes from eukaryotes are inserted into existing plasmids from

prokaryotes in order to transform them, theoretically the bacteria would be eager to accept them.

However, it could be possible that there are some unknown markers that distinguish a plasmid

that has been “tampered” with an untouched plasmid, which would explain the need for a

complicated procedure in order for transformation to occur. One possible “marker” that could

distinguish between “tampered” and regular DNA could be similar to restriction modification,

which is the addition of methyl groups to the bacteria’s own DNA to distinguish it from

bacteriophage DNA. This presents another area of study to be investigated: how exactly do

bacteria respond to their environment and can they distinguish between modified and untouched

plasmids? If there is a distinction, then finding a way to minimize the differences between

naturally occurring plasmids and modified plasmids would significantly simplify the procedure

and produce more consistent results.

References

Bacterial Transformation Lab . Bacterial Transformation Lab Slide Show. (n.d.).

https://docs.google.com/presentation/d/1UE8AAHIryJnmqTqu919iuo03Gz3bWIfuWducEDTO6

ak/edit#slide=id.p
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Griffith’s experiment. Griffith’s Experiment - an overview | ScienceDirect Topics. (n.d.).

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/griffiths-exp

eriment#:~:text=In%201928%2C%20in%20an%20attempt,function%20of%20a%20bacterium%

20changes.

MiniOne. (n.d.). M6300 Ley it Glow Bacterial Transformation MiniLab Student Guide.

https://www.google.com/drive/download/

Riggs, A. D. (2021, May 25). Making, cloning, and the expression of human insulin

genes in bacteria: The path to Humulin. Endocrine reviews.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152450/#:~:text=As%20pictured%2C%202%2

0E.,methionine%20to%20an%20insulin%20tail.