1. Reed Woyda, Shannon Helmer, Molecular Biology Biol 479/579 Class 2016, Allison M. Land
Minnesota State University, Mankato, Department of Biological Sciences
Abstract
References
Conclusions
Victor Frankenstein was one of the first to introduce the idea of
modifying an organism in a novel written by Mary Shelley in 1818.
This study presents a way of modifying an organism which is much
less gruesome than Frankenstein’s creation, but produces visually
stimulating results. The goal of this experiment was to insert the
green fluorescent protein (GFP) into both Escherichia Coli cells
and human embryonic kidney 239T cells. It is hypothesized that
introduction of the GFP gene will cause visually detectable changes
as a result of genomic changes. Insertion of the GFP into a plasmid
was performed using restriction digests, polymerase chain reaction
(PCR), gel electrophoresis, gel purification, transfection and
ligation. Verification of introduction of GFP into plasmids was
done by means of gel electrophoresis and insertion into mammalian
cells with immunoblotting. Frankenstein’s monster may or may not
have been created with the best of intentions, but the experiments
performed here are instrumental tools in disease discovery,
classification and prevention, and are key in understanding the
mysteries bodies we live in.
Background
GFP was originally aquired from Aequorea victoria which is a
jellyfish of the class hydrozoa. GFP is routinely used as a genetic
marker in order to verify introduction of a new products or features
into genetic material. The fact that GFP can be both introduced to
prokaryote and eukaryotic cells also adds to its desirability in
molecular biology.
Introducing Green Fluorescence Into Homo sapiens And Escherichia Coli Cells
Figure 1 GFP Induction by L-arabinose. E. coli cells
were transformed with pGLO plasmid containing GFP
(27kD) under the control of the arabinose promoter.
Lanes indicate times samples were taken, in hours.
SDS-PAGE with a coomassie stain was used to
express total protein (a). Proteins separated by SDS-
PAGE transferred were to membrane (b) via mouse
and goat antibodies (c).
c
Methodology
pGLO plasmids were transformed into E. coli via heat shock using a CaCl2 solution. GFP is under the control of the araBAD promoter within the
pGLO plasmid. Transformed E. coli were then grown on plates containing various combinations of LB, Ampicillin, and arabinose. GFP induction
my L-arabinose was measured using optical density as a means of adjusting sample volumes for comparison of protein levels. Samples were taken at
different time intervals and validity of protein expression was measured by means of SDS-PAGE with coomassie staining and immunobloting. DNA
was purified from E. coli cultures and transformed with pGLO using a miniprep protocol. Addition of restriction sites to the termini of the purified
DNA was performed using PCR and subsequently verified using agarose gel electrophoresis. DNA from the PCR was then cleaned up to remove
unused primers. Restriction digests were performed on both the cleaned up PCR product and the pcDNA vector using KpnI and EcoRI restriction
enzymes. Digests were then run on agarose gel to separate the cut DNA. Cut DNA segments were then removed from the agarose gel and purified
using a salt buffer with the addition of heat. Purified cut DNA segments, GFP and pcDNA, were then ligated together using T4 DNA ligase from a
bacteriophage. Ligated plasmid DNA was then transformed into CaCl2 competent E. coli cells using heat shock. Transformed cells were then plated
on LB-Amp or LB for verification of ligation and transformation. Successfully ligated colonies were then cultured overnight in a 37 ̊C water bath.
DNA was purified from transformed E. coli cultures, containing ligated pcDNA, using a plasmid miniprep protocol. Verification of successful
ligation was done using restricting digest, using the same restriction enzymes discussed previously, and subsequently run on agarose gel
electrophoresis. 239T cells transfected with pcDNA-GFP, pGLO and pcDNA were used to create whole cell lysates. Cell lysates were then run on
polyacrylamide gel and transferred to a membrane for immunoblotting.
Figure 4 Transformation of sticky
end plasmids into CaCl2 competent
E. coli cells. Purified GFP (lane 1),
PCR water (lane 2), pGLO plasmid
(lane 3,5) and solely CaCl2 cells run
on agarose gel.
Figure 3 Restriction Digest
of Purified GFP PCR
Product and pcDNA with
KpnI-HF and EcoRI-HF.
Bands separated via agarose
gel electrophoresis.
a b
Figure 2 Insertion of restriction
cut sites via PCR. DNA from E.
coli cells transformed with the
pGLO plasmid, containing GFP,
was isolated. Restriction sites
were added via PCR and run on
agarose gel using electrophoresis
and imaged under UV.
As hypothesized, GFP was successfully inserted into both E. coli and 239T
cells. As seen in figure 1, GFP was expressed, under the control of L-
arabinose, after transformed into E. coli, having peak expression at 3
hours. Addition of restriction sites to the termini of the GFP gene was
successful, verified by visible bands at 0.08kb in figure 2. After digestion
of the pcDNA and GFP with KpnI and EcoRI we find bands at 6.0kb and
0.7kb, corresponding to pcDNA and GFP respectively (figure 3).
Unfortunately purified DNA from gel electrophoresis was unsuccessfully
ligated into pcDNA as shown in figure 4.0, thought to be due to
contamination with nucleases. Additionally, immunoblot of cell lysates of
239T cells (figure not shown) was not able to be determined due to
technique error which caused a poor image to be produced.
A. Land. 2016. Molecular Biology 479 Lab Manual.
Results