Ryan Hahn

Biology 302 AA
GFP Transformation and Selective Activation in Escherichia Coli

Introduction
As medical research advances, scientists have found ways to treat a range of diseases that affect humans
including diabetes. Using plasmid transformations containing the recombinant DNA for human insulin in
Escherichia Coli, scientists are able to manufacture insulin on a large scale for patients1. The
manufacturing of insulin in Escherichia Coli is cost-effective, manageable, productive, and efficient.
However, production has drawbacks including improper protein folding, an absence of post-translation
modifications, and plasmid loss1. In the Escherichia Coli, the use of GFP can be used to demonstrate
successful plasmid transformation confirming the presence of the insulin gene in bacterial colonies and
has applications for quantifying protein production under the same gene promoter4.

GFP or Green Fluorescent Protein is a protein that emits wavelengths in the green area of the visible
emission spectrum. GFP has two main applications for monitoring gene expression: fusion tagging and
transcription reporting2. The latter application uses GFP as a reporter gene that is expressed under a
common gene promoter found in Escherichia coli demonstrating that the transformed gene is expressed in
correlation with GFP expression3.

In this experiment, we wanted to identify which sugar media(s) induce GFP production in Escherichia
coli after a plasmid transformation containing GFP and bla, a gene encoding the beta-lactamase protein
that digests ampicillin antibiotic4. Differential GFP production is confirmed using SDS-PAGE and
quantified by spectrophotometric analysis of each sample.

Results
Phenotypic Presentation of Escherichia coli under UV Light

Escherichia coli Observed under UV Light Following Transformation, Growth in Different Sugar Medias
Ryan Hahn
Biology 302 AA
Figure 1: The top three photos are Escherichia coli colonies grown in LB/ampicillin media with fructose, lactose,
and arabinose respectively. The remaining photos are colonies with LB/ampicillin media, LB/ampicillin/arabinose
media with professional cells, and LB/ampicillin media with professional plasmid. Colonies that glow under UV
lighting demonstrate GFP expression. The arabinose colonies glow the brightest, demonstrating higher levels of
expression for GFP than any other sugar media.
The presence of Escherichia coli colonies grown in an antibiotic media demonstrates a successful
selection and transformation of the GFP+bla plasmid. Fluorescence intensity under UV indicates GFP
expression. Comparing phenotypic presentation based on observations, arabinose sugar media induces
GFP transcription at higher levels than fructose or lactose (Figure 1). Observational data alone does not
conclude that the fluorescence is derived from GFP or the levels of expression for GFP, requiring SDS-
PAGE to confirm the expression of GFP in conjunction and the use of spectrometry to measure the levels
of GFP production.

GFP Screening in E. coli

Potential GFP Expression Confirmed via SDS-PAGE in Different Escherichia coli Treatments

Figure 2: Following cell lysing using SDS solution, the following protein bands were detected on an SDS-PAGE gel.
The arabinose and professional cells solutions all share sharp bands above the 25kDa marker, indicating high
levels of GFP expression. The remaining solutions have faint bands suggesting GFP had low expressions in their
cells or non-GFP proteins of the same size were present at low levels.
Following cell lysing and protein denaturing, the following protein bands detected confirmed in
conjunction with observations using UV light the high levels of expression of GFP in arabinose media
and professional cells. Each solution had sharp bands above the 25kDa; GFP is 27kDa in size (Figure 2).
Lactose, fructose, professional plasmid, and LB/ampicillin had fainter bands above the 25kDa marker
suggesting detection of proteins greater in size relative to GFP and of a different identity. SDS-PAGE
data alone only suggests the presence of a protein with a similar size to GFP in cells grown in arabinose
media; it does not conclusively identify GFP as the protein detected.
Ryan Hahn
Biology 302 AA

Differential GFP Production in E. coli Growth Medias

70000
60000
GFP emission (485 nm)

50000
40000
30000
20000
10000
0
1 2 3 4 6 8
Growth Media Treatments

Top Blue Table AA Class Average

Figure 3: The following chart shows different GFP production levels in Escherichia coli colonies in the following
growth medias in the lab group versus the class average: LB+amp. (1), LB+amp.+arabinose (2),LB+amp.+fructose
(3), LB+amp.+lactose (4), LB+amp.+ arabinose professional cells (6), and LB+amp.+professional plasmid (8).
After cell lysing using lysozyme to preserve GFP function, the cell solutions were measured by a spectrophotometer
for fluorescence intensity. Higher intensity indicates higher levels of GFP activity.
Using spectrophotometry, GFP expression measured to be qualitatively higher in arabinose media than
other sugar media (Figure 3). Cultures with GFP had higher emission data values, indicating that the
solution was more fluorescent. Data confirmed phenotypic comparisons between treatments. Class
averages for each treatment were higher for nearly all treatments but the group measurements followed
the same trends as the class averages showing internal consistency.

Conclusion
GFP has a wide range of applications that allow for the confirmation of bacterial transformations.
Phenotypic observations between different growth media alone are inconclusive alone but produce trends
in unempirical physical measurements and characteristics. SDS-PAGE when compared with UV light
observations confirmed the presence of GFP in cells and spectrophotometry measured the levels of GFP
expression in each treatment. Synthesizing data from each protocol, it is concluded that arabinose sugar
induces the transcription of GFP. This indicates that the GFP gene is under the control of the arabinose
promoter. Lactose and fructose show levels of expression similar to the base level of the LB/AMP media
with no sugar substrate, indicating these sugars are not able to induce significant levels of GFP
expression. Due to the availability and versatility of GFP as a reporter gene, future medical research may
be able to utilize GFP to research diseases whose genetic origins still puzzle scientists.

References
Ryan Hahn
Biology 302 AA
1: Baeshen, Nabih A., Mohammed N. Baeshen, Abdullah Sheikh, Roop S. Bora, Mohamed Morsi M
Ahmed, Hassan A I Ramadan, Kulvinder Singh Saini, and Elrashdy M. Redwan. "Cell factories for
insulin production." Microbial Cell Factories 13, no. 1 (2014). doi:10.1186/s12934-014-0141-0.
2: Patrick, Marcy. "Plasmids 101: Green Fluorescent Protein (GFP)." Plasmids 101: Green Fluorescent
Protein (GFP). Accessed February 04, 2017. http://blog.addgene.org/plasmids-101-green-fluorescent-
protein-gfp.
3: Alberts, Bruce, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Molecular Biology of the Cell. 6th ed. New York: Garland Science, 2015.
4: Martin-Morris, Linda. Laboratory Manual Biology 302. Winter 2017. Seattle, WA: Professional Copy
Print, 2017.