The document discusses the green fluorescent protein (GFP) discovered in the jellyfish Aequorea victoria. GFP causes the jellyfish to glow green through bioluminescence. In the 1960s, Osamu Shimomura isolated GFP from Aequorea victoria and his work helped uncover how GFP fluorescence works. GFP has since become an important tool in molecular and cell biology by allowing researchers to track cellular movements and protein expressions through genetic tagging with the GFP gene.

1. Pglo Plasmase
Abstract
When trying to transfer several specific plasmids into a new host DNA, you need a way to check
and see if the desired plasmids were combined to the DNA, that's where there pGLO test comes in.
By using an ultraviolet light, a person is able to immediately see if the new plasmids have been
accepted into the bacteria by whether or not a bright green fluorescent light is emitting from the
bacteria. So the process in how pGLO is expressed would be the plasmid itself is extracted from the
A. Victoria, modified and inserted into bacteria with the three genes. The beta lactamase then comes
first, to give it a resistant to the ampicillin antibiotics that would initially kill it. This then allowed
the araC operon to take the arabinose and allow ... Show more content on Helpwriting.net ...
Then label each of them either pGLO positive and pGLO negative, depending on which one took
the plasmid (4). Place both test tubes on micro–centrifuge on ice for ten minutes. Label the agar
plates with three for pGLO positive and three for pGLO negative. Label the six agar plates with
what they are, whether it is just LB, LB and ampicillin, or LB, ampicillin, and araC (4). There
should be one for each positive and negative. After the ten minutes in the centrifuge has passed, take
them out and place them in a forty two degrees Celsius heating block for fifty seconds. Once the
fifty seconds have passed, transfer the tubes back to ice for two minutes. Take the micropipette and
begin adding pGLO positive or pGLO negative to the correctly labeled plates. Once the pGLO is
added to the agar, use the spreader to spread out the culture until it is dry on the agar plate. Make
sure that before and after every use of the spreader that it is dipped in ethanol and sterilized with the
Bunsen burner, to avoid contamination. Once all of the bacteria is dried onto the agar plates, use
Para film to seal the agar cultures shut. Allow for it to incubate at thirty seven degrees Celsius for
twenty four hours(4). After time has passed, check for growth and use an ultraviolet light to check
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3. The Evolution Of The Green Fluorescent Protein ( GFP )
No other aquatic organism like the jellyfish instills fears in ocean swimmers. Known for their
graceful movements, tentacles, and stinging ability, the jellyfish is avoided at all cost. There is,
however, a particular species of jellyfish Aequorea Victoria that has been very beneficial to
scientists. This species of jellyfish produces green bioluminescence from small photo organs located
on its umbrella. In the 1960, while working at Princeton University, the scientist Osamu Shimomura
was attempting to answer the question how some organisms can produce light which led him to
work on the jellyfish and the discovery of GFP. In his work with the jellyfish Aequorea Victoria, he
discovered and isolated Aequorin, the bioluminescence protein. ... Show more content on
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As part of his work, he succeeded in varying the color of the light emitted by GFP so that different
proteins and multiple, simultaneous biological processes could be tracked using GFP. His work that
provided a better understanding of the fluorescence properties of GFP and its' application as a
marker. (Ehrenberg, 2008) In 2008, Osamu Shimomura, Martin Chalfie, and Roger Tsien were
awarded the Nobel Prize in Chemistry in 2008. This research and that conducted by other scientists
transformed the ability of the scientific community to track cellular movements of diseases such as
cancer by using GFP as an intracellular marker. Using a gene that carries instructions to make GFP,
scientists can attach glow–in–the–dark tags to selected proteins, either in cells in lab dishes or inside
living creatures, to track their expressions and localization. (MacLachlan, 2011). One use of GFP is
as a component of the plasmid pGLO. pGLO is made through recombinant DNA technology and
consists of the GFP gene that codes for green fluorescence, a gene (bla) that codes for the enzyme
beta–lactamase which produces bacteria resistance to the beta–lactam family of antibiotics of which
ampicillin is one such antibiotic and lastly AraC which binds the arabinose thereby regulating
expression of GFP. GFP will not be expressed unless the carbohydrate arabinose is present. Plasmid
is small pieces of DNA that is distinct from a cell's chromosomal
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5. Agarose Gel Results
The following figure shows the agarose gel results of the digested DNA plasmids 1 and 2 as well as
the molecular weight markers. This allows for the visualisation of DNA by adding fluorescent dye
SYBR Green to the gel loading dye which was then added to the restriction digest. These results
belong to Jessica and Vince in Group 13. My results did not provide clear bands above the top
maker, which may be a result of the restriction digest not working properly. However, these results
below illustrate two clear bands of each of the digested plasmids and the bands are below the top
maker.
DNA plasmid 1 is determined to be Integrin insert since the size of plasmid fragment is 1122 bp,
which is relatively close to 1,250 bp, as seen in Figure 4. ... Show more content on Helpwriting.net
...
The product of the restriction enzyme digestion of plasmid DNA was analysed visually on agarose
electrophoresis. The identity of the plasmid fragment in plasmid 1 at 1122 bp is established to be
Integrin. The identity of the plasmid fragment in plasmid 2 at 501 bp is determined to be VEGF.
The isolated DNA plasmid is quantitated using UV absorbance and SYBR Green fluorescent
methods. The concentration of plasmids 1 and 2, using UV absorbance, was established to be 3227
µg/mL and 3951 µg/mL, respectively. This method also determines the 260/280 ratio for plasmid 1
which was 1.390 and for plasmid 2 which was 1.342. Furthermore, the plasmids are both
contaminated with proteins. The SYBR Green fluorescent method is more accurate than the UV
absorbance method since it is more specific for double–stranded DNA molecules. Therefore, the
SYBR Green assay results are lower than those obtained in the UV absorbance and are estimated to
be 163 µg/mL and 202 µg/mL,
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7. Notes On Green Fluorescent Proteins
1. Green Fluorescent Protein (GFP) GFP is a widely used tool in the field of Molecular biology and
Cell biology. It involves emission of fluorescence under Ultra Violet light which allows for direct
investigation into the inner working of cells. Green Fluorescent Protein was first isolated from the
jelly fish Aequorea victoria by Osamu Shimomura (Shimomura et al., 1962). Since its discovery, it
has become useful in the field of science. The GFP chromophore is formed from tri peptide in the
primary structure of GFP. Its fluorescence is turned on when exposed to molecular oxygen. The gene
of GFP has been introduced into many bacteria, yeast, fungi, plants and humans (Amsterdam, A. et
al., 1996) and it is still gaining rapid ground in the field of biological science.
2. Structure of the Green Fluorescent Protein
The GFP is composed of two structures; a barrel beta structure consisting of 11 beta strands and an
alpha helix containing the covalently bonded chromophore 4–p–(hydroxybenzylidene)
imidazolidin–5–one. The chromophore is in the centre of the barrel beta structure (Tsien, 1998). The
size of the beta structure is 42Å long and 24Å in diameter. Due to its length and width it creates a
beta like structure which is common in the GFP family (Yang, 1996). The chromophore is made
from a tri–peptide Ser65–Tyr66–Gly67. To form its structure GFP first folds into a nearly native
conformation, then the imidazolinoneis is formed by nucleophilic attack of the amide of Gly67 on
the
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9. The Invention Of The Electron Microscope By Ernst Ruska...
In the scientific community, there have been many advancements in the technologies and processes
used to help us understand the truth of the world around us. The invention of the electron
microscope by Ernst Ruska and Max Knoll in 1933 is just an example of one of these advancements.
There is another advancement that has roots over one hundred and sixty million years old, and that
is the green fluorescent protein (GFP). The fluorescence from GFP molecules has made it feasible to
look through a microscope at a spatial resolution higher than the diffraction limit. The green
fluorescent protein was first found in the Aequorea Victoria, or the crystal jellyfish. It is a
bioluminescent hydrozoan jellyfish, or hydromedusa (1). In the crystal jellyfish, there are two
proteins that are responsible for its bioluminescent, aequorin and GFP. They are both located in the
photo–organs of the jellyfish. The jellyfish releases calcium ions which the aequorin binds to. The
process of binding gives off a blue light, which is then absorbed by the GFP. The blue light absorbed
is then converted into the green light that GFP is named for (2). At the moment there is no apparent
reason for the bioluminescence displayed by the Aequorea Victoria since it has not been observed
being used as a defense mechanism or as a signalling mechanism. Osamu Shimomura was the first
person to isolate the GFP. He started to study the bioluminescence of crystal jellyfish at Princeton
University in 1960 and he and
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11. Aequorea Research Papers
Aequorea victoria is a jellyfish in Puget Sound, Washington State. The small hydromedusa grows to
be 5 to 10 centimeters in diameter and live for around six months. The tiny jellyfish is most
famously known for the luminescent protein aequorin and the fluorescent molecule GFP or green
fluorescent protein (Mills 1999). These two proteins have proven to be quite useful in biochemistry
and biomedical research. More commonly used is green fluorescent protein, which has been used as
a marker for gene expression in various types of research. Green fluorescent protein was first
recognized in the early 1960s by a group of researchers studying the bioluminescent properties of
the Aequorea victoria jellyfish. They discovered that the jellyfish produces green bioluminescence
from small photoorgans located on its umbrella. Osamu Shimomura found that in order to
bioluminesce Aequorea releases calcium ions. These bind to a protein that he called aequorin, which
release blue light upon calcium binding. The blue light is absorbed by green fluorescent protein,
which in turn gives off the green light (Shimomura, O., Johnson, F. H. and Saiga, Y. 1962.) In 1987,
Douglas Prasher was the first person to recognize the potential for Green fluorescent protein to be
utilized as a tracer molecule. Due to proteins being extremely small and unseen even under electron
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With the aid of GFP, researchers have developed ways to watch processes that were previously
invisible, such as the development of nerve cells in the brain or how cancer cells spread. Tens of
thousands of different proteins reside in a living organism, controlling important chemical processes
in minute detail. If this protein machinery malfunctions, illness and disease often follow. That is
why it has been imperative for bioscience to map the role of different proteins in the body. "(The
Nobel Prize in Chemistry 2008 – Press
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13. Green Fluorescent Protein Lab Report
1. The technique that allows scientists to follow with their own eyes the dynamic movements of
specific proteins as they occur within the living cell is the green fluorescent protein. This is a protein
that is extracted from only a few jellyfish. This diffuses a green fluorescent colored light. The green
fluorescent protein can be blended to the protein and can work normally and so does the protein
itself that it is binded to. The protein is not affected and can be moved and transported throughout
the cell and can then be seen.
2. The early endosomes progress gradually. The early endosomes transform into the late endosomes
because of the lowered levels of ph, when Rab proteins are traded, and the earlier endosomes
internal structure has a crucial ... Show more content on Helpwriting.net ...
A big part of the gene's segment is not shown or present in the mRNA or the cDNA that is created
from it. This means that DNA is taken out from the primary gene transcript while the pre–mRNA
was being prepared.
7. The properties of RNA that have convinced investigators that it may have played an important
role in the evolution of life in earth is that RNA is able to encrypt information and is able to
assemble chemical reactions. Before ribozymes were discovered, proteins were only able to catalyze
and only nucleic acids encrypted information. Obviously, RNA can do both and since the discovery
of it, many scientists now believe RNA played an important role in the evolution of life.
8. The enzyme must correctly work and then it will make the chromosome telomere shorter and will
gradually top the cell division in some of the cells. This would reverse the diseased condition and
then turn back into the normal state it was in before.
9. When experimenting, normal strain of the bacteria creates relatively low amounts of beta–
galactosidase. With the mutant strain, the operating site is changed and then has the constitutive
synthesis of the higher amounts of the beta–galactosidase when the lactose is available or not
available. Lac + is when lactose is used for carbon and Lac– for the mutant, is used when lactose is
not available to
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15. Fluorescent Tagged Protein Research Paper
Essay 47: Fluorescent Tagged Proteins
Proteins tagged with fluorescent markers are extremely versatile tools in cell and molecular biology
research. A fluorescent marker is, as the name suggests, a molecule that emits light of a particular
wavelength following exposure to a photons of a shorter wavelength, for example a laser beam. If a
fluorescent marker is a reasonable size and chemically stable enough to be attached to a protein, it is
potentially useful to molecular biologists. In many cases, the proteins in question are monoclonal
antibodies generated against a particular target of interest such as a cell surface receptor, a
cytoskeletal protein, or a cellular organelle. In some cases, a fluorescent label is used to track the
fate of a particular protein as it is processed in the ER and Golgi complex, transported along
microtubules, or secreted from the cell altogether. In still other cases, two or more fluorescently
tagged proteins are introduced into the same cell to see whether they localize in the same or separate
compartments or organelles. ... Show more content on Helpwriting.net ...
In this method, cells are incubated with antibodies conjugated with fluorescent dyes such as FITC
(fluoroscein isothiocyanate), which emits green wavelengths of light; PE (phycoerythrin), or Texas
Red, both of which emit red wavelengths of light. Newer fluorescent dyes emit at yellow and blue
wavelengths as
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17. The Importance Of Green Fluorescent Protein
In the 1960's, Green Fluorescent Protein was discovered to be responsible for Aequorea victoria's
fluorescence under UV light (Niwa et al., 1996). Today, GFP is often used in protein tagging and has
made it possible for scientists to study expression and track proteins in vivo. With the rising
importance of GFP fusion proteins and other recombinant vectors, the metal affinity of Histidine
helps to make the protein purification process easier (Lilius et al., 1991). His tags are commonly
used to purify proteins through immobilized metal–affinity chromatography (IMAC) (Lilius et al.,
1991). This rapid and efficient method separates the recombinant protein from unwanted products
such as RNA. The His tag DNA sequence is inserted into the ... Show more content on
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It is of strong interest to molecular biologists because of the strong light emission of 508 nm under
UV light. UV wavelengths excite the fluorophore, contained in the Ser – Tyr – Gly sequence near
GFP's N–terminus, and a bright green fluorescence is given off as a result (Niwa et al., 1996). GFP
can be successfully tagged to many proteins without disturbing their function (Feilmeier et al.,
2000). This allows scientists to exploit the protein for its usefulness in marking proteins in vivo.
GFP tagging has many applications. It can be used to study gene expression by inserting gfp after a
promotor in the plasmid, so that if expressed, the colony expressing the gene of interest can be
identified by a green fluorescence of GFP under UV light (Feilmeier et al., 2000). Specific cellular
organelles can be tagged, such as the endoplasmic reticulum, to visualize protein localization and
networking inside the living cell. GFP can also be used to monitor cofactor levels within a cell in
real time. For example, when GFP was tagged to proteins that contain a receptor sensitive to Ca2+,
they interacted in a way so that the fluorescence of the fusion protein was proportional to the
calcium ion concentration with less than a 1 second lag in the cell (Romoser et al., 1997).
Throughout the decades, GFP mutants have been created to give brighter emission and different
wavelengths. Researchers can track separate cell pathways at the same time, since different proteins
can
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19. Nanotechnology In Michael Grant's Bug Man
BZRK, a novel by Michael Grant, is a science fiction story, about the misuse of nanotechnology. In
the novel BZRK, nanotechnology is used for negative reasons, by the Armstrong Fancy Gift
Corporation. This is an analogy of a world with nanotechnology in a modern day era. The
technology developed the ability to make microscopic robots that can enter the human body through
natural openings to change structures at a molecular level. They created it for medical purposes, for
example, "to send his tiny minions in to kill cancer cells" (Grant, 72). Nanotechnology is being
negatively used to take away freedom and chose for human beings. This good technology is used the
wrong way to control the human mind and commit murder. Many of people with access ... Show
more content on Helpwriting.net ...
Burnofsky, a sixty year old man, works for Armstrong Fancy Gifts Corporation as a twitcher. The
twitchers manipulate nanobots deep inside the human brain. He tries to explain the reality of what
they are doing to Anthony, after the McLure plane crash. Anthony is a teenager, also known as the
Bug Man. "Anthony, you're a mass murderer now, up in the macro, not just shooting spiders down in
the meat" (Grant, 30). The Bug Man ignores the comment, but Burnofsky says "you'll grow up
someday and realize what you did" (Grant, 31). Burnofsky knows the price they are paying by
killing humans in order to get "one step closer to a world of perfect peace, happiness and universal
brotherhood" (Grant, 31). A twitcher One–up, only seventeen years old was given the job to kill
Bailey, a UN employee. Burnofsky stated "a seventeen–year old girl should have some deniability
for murder" (Grant, 126). Burnofsky notes that "Twenty–seven twitchers to take over the world.
Half of them nothing but messed–up children" (Grant, 127). Even Plath wanted to commit murder,
but Keats talks her out of it. "Plath was asking Keats if she should kill Benjamin Armstrong" (Grant,
384). Keats responds "We're not them" (Grant, 385). Teenagers with troubled pasts are playing God
by having control over life and death by using the
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21. Fluorescent Protein Coding Regions Of Species Of The...
Fluorescent Protein coding regions of species of the Cnidarian phyla, will be subjected to an
evolutionary analysis focusing on gene duplication, expansion and divergence. This will allow the
evolutionary processes that led to the development of these gene become clear. Changes due to
mutations and other factors which occurr in DNA sequences cause variation of genes over
evolutionary time. Characterisation of fluorescent proteins across sister phyla will allow accurate
conclusion of the origins of such proteins. It is suspected that gene expansion across evolutionary
time resulted in the diversification of the fluorescent genes to exhibit a vast array of colours seen
across today's Cnidaria. One phyla of particular interest in the Cnidarians, with their vast colour
array of their fluorophores. The results were inconclusive and the hypothesis was not answered.
Key Words: Cnidarian, Zoanthid, green fluorescent protein, evolution, gene expansion
Introduction
The first thing to note is luminescence is a generalized term describing the production of visual light
with a lack of radiant heat termed "cold light". Three main categories describe the types of
luminescence (please refer to Table 1); biological, chemical and physical. Bioluminescence is
another broad term defining any form of luminescence produced by organisms, predominantly seen
in marine species. This phenomena is seen in some species of cephalopods of the Tesuthida (squid)
order and many of the members of the
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23. The Transformation Of Bacteria ( Green Fluorescent Protein )
Transformation in bacteria is something that could be essential for survival in a bacteria. In order to
perform this transformation naturally a bacterium must considered competent, otherwise it must
undergo an artificial transformation. Being a competent cell means that the bacteria can take up
DNA from its environment naturally (5). Those that are not competent such as Escherichia coli that
are not naturally competent can be tested with an artificial transformation, such as what we will use
in this experiment. Methods used can obtain things such as chemical mutagens or radiation (1). The
gene used for the transfer is the GFP gene (Green Fluorescent Protein), which gives an illuminating
appearance under a UV light when conducted properly, ... Show more content on Helpwriting.net ...
Experiments introduce the idea that the uptake of DNA by competent bacteria can be the result for
survival reasons such as need for food or evolution purposes (5). The difference between natural and
artificial gene transformation in a bacteria, however, with those who are incompetent versus
competent allow he or she to conduct an experiment with artificial transformation. By doing this one
may be able to determine and observe the formation of biofilms or mutations by transferring a gene
to another organism for beneficial or harmful purpose. This could lead to finding resistants or if a
gene introduced has any effect on the bacteria. In the experiment, the pGLO plasmid contained
encodes the gene for GFP as well as a gene for resistance to an antibiotic allowing a transformation
to take place when adding a carbohydrate such as arabinose to the medium. Research has been
found on the horizontal gene transfer on how virulence factors are acquired as well as genes
resistant to antibiotics spread with microorganisms (5). By conducting a similar experiment he or
she can observe similar findings or observe the results and conduct some sort of understanding of
these ideas. The findings in this experiment are to show a positive result in what causes growth and
the glowing when the plasmid pGLO is added to the positive as well as the difference in the
medium. Understanding the way artificial
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25. A Study On Green Fluorescent Proteins
Question:
Would the GFP gene in a pGLO transformed E. coli be activated by a sugar other than arabinose?
Introduction:
Green Fluorescent Protein, produced by the bioluminescent jellyfish Aequorea victoria, is a protein
that fluoresces green under ultraviolet light. Since its discovery, properties of the protein have been
improved by mutations in the gene resulting in the expansion of its spectrum, which now contains
brighter variants and multiple different colors. GFP is used in a wide variety of applications and
technologies. Its many different applications have contributed greatly, and continue to do so, in
numerous fields of study including, but not limited to, cellular and molecular biology, microbiology,
biotechnology, and medicine.
In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding
green fluorescent protein by inserting a plasmid containing the GFP gene, beta–lactamase, and
arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of
ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible
for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.
The requirement of the presence of arabinose in the pGLO lab in order to activate the gene for the
green fluorescent protein highly intrigued me. The idea that a simple sugar held the key to the final
step of the transformation, made me wonder if other sugars,
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27. Zebrafish Experiment
Introduction
Zebrafish (Danio rerio) are a popular freshwater fish that have been modified by researchers to
produce several transgenic strains. The strain of transgenic zebrafish that express fluorescent colors
have been given the name Glofish, for they contain different fluorescent proteins such as Green
Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), Blue
Fluorescent Protein (BFP), or Purple Fluorescent Protein (PFP) (Vick, B. M., A. Pollak, C. Welsh, J.
O. Liang, 2012). There are several benefits of using Glofish as a model to portray and understand
inheritance patterns of transgenic genes. Glofish are small size in size, produce a high number of
progeny, have similar genetic control to humans, ... Show more content on Helpwriting.net ...
Phenotypes pertaining to fins, fin length, and color were observed. Only a few phenotypes, such as
fin length and presence of specific fluorescent proteins, were decided on to be important in
understanding the genetics (Biology 2202 Genetics Laboratory Handout, University of Minnesota –
Duluth, 2015). Fishes with these phenotypes were counted and recorded.
Generating Hypotheses
Numbers of the wild–type and transgenic fish were compared. Type of transgenes and type of
inheritance were hypothesized based on the patterns seen in the offspring. Using that information
can help determine the parent genotypes and thus determine a final inheritance pattern. A null
hypothesis was then formed relative to the genes and inheritance pattern of each tank (Biology 2202
Genetics Laboratory Handout, University of Minnesota – Duluth, 2015).
Data Analysis
Hypothesis were generated by comparing the phenotypes of each tank. The hypothesis included the
ratio and expected inheritance pattern, and that any difference is due to chance. Using the
information collected based on the genotypes of the parents and the formed hypothesis, the expected
value for each phenotype were calculated and recorded. A chi–square test was used to determine the
P–value and conclude the generated
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29. Lab Report On Pglo Transformation
Genetic transformation is a process that occurs when the DNA of a cell is altered by the introduction
of genetic material from a different source, like from another cell or a virus. This process was
discovered in 1928 by Fred Griffith while he was performing an experiment where he injected
different combinations of live and dead strains of Pneumococcus bacteria that was either virulent or
non virulent into mice. When only the dead virulent strain was injected, the mice were unaffected,
but when both the dead virulent and the live non virulent strains were injected, the mice died. This
occurred because an instructional agent passed between the living and dead bacteria, causing the
DNA of the living non virulent strain to be transformed and produce ... Show more content on
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coli) with a pGLO plasmid, which will cause it to take in and express the Green Fluorescent Protein
(GFP) gene. This gene, which has been encoded into the pGLO plasmid, is found in jellyfish and
causes them to produce the protein that makes them fluorescent green. A plasmid is a circular piece
of DNA found within a prokaryotic that contains certain genes for a trait that can help the
prokaryote to survive. They can also be extracted from prokaryotes and modified to express a
desired gene. Bacteria naturally exchange plasmids, causing them to gain traits from the other and
increase their chance of survival. To integrate plasmids into DNA, restriction enzymes cut the DNA
at certain points, creating "sticky ends". Sticky ends are areas of unmatched nucleotide bases that
can be attached to corresponding nucleotide bases. Once the DNA and plasmid have been cut, the
sticky ends from both are attached together so that their nucleotide bases match and are sealed with
the enzyme ligase. After this, the cell can express the gene that the plasmid contains. If the E. coli
bacteria successfully take in the plasmid containing the GFP gene, they will express the protein and
become fluorescent green. The pGLO plasmid also contains a gene called beta–lactamase that
causes resistance towards the antibiotic ampicillin. Therefore, if the E. coli bacteria successfully
take in the plasmid, they will
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31. The Transformation Of Genes On The Mitochondrial And...
Transformation is a procedure where cells take up foreign DNA from their environment. This can
happen in either prokaryotic or eukaryotic cells. A circular self–replicating molecule of DNA known
as a plasmid transfers genetic information between bacteria enabling the microbes to gain antibiotic
resistance as well as the ability to adapt to a new environment. Bacteria also have the capability to
acquire small pieces (at random) of DNA from their environment but this however is said to be
inefficient because only a small fraction of cells will uptake DNA and become transformed.
Expressions of genes on the plasmid will alter the genotype and phenotype of the cell. Under
specific laboratory conditions a cell which is incubated with the plasmid DNA has the ability to
absorb the plasmid into its own cytoplasm. Because these natural processes can potentially be
modified by scientists, the transformation procedure can be used as a means of treating human
diseases to increase the quality of life.
Genetic transformation will be assisted with the use of plasmid pGLO which contains four features.
Origin of replication (ori), ampicillin resistance gene (bla), arabinose promoter protein (araC) and
the Green Fluorescent Protein (GFP gene). The Green Fluorescent Protein (GFP) is extracted from a
bioluminescent (encodes fluorescent protein) jellyfish called Aequorea Victoria which glows green
when exposed to ultraviolet light. GFP holds many advantages due to its stability and the fact
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33. Pglo Transformation Lab
Shaddi Martinez
Ms. Herrera
LSC 348 Lab
12/3/16
pGLO Transformation: A Look into Our Future with DNA and Genetics
Abstract
This lab investigated the central dogma of molecular biology by studying the expression of jellyfish
Green Fluorescent Protein in E. coli (Mardigan, 2011). After transforming E. coli with the GFP gene
under the arabinose operon promoter, GFP expression was analyzed with and without arabinose.
Induction was seen only in media with arabinose. To further study GFP protein and structure, an
SDS–Page or gel electrophoresis was conducted where proteins were charged to allow them to move
in an electrical field in terms of their size. To further study the role of the arabinose operon in GFP
expression an overnight culture was done where the plate was frozen and incubated to induce
optimal protein folding. The GFP was then purified. Centrifugation, use of buffers, and a column
was used to separate the GFP and bacterial cells.
Introduction ... Show more content on Helpwriting.net ...
coli the bacteria that will be used to house the GFP is the competent cell. They are called competent
cells because they are able to accept foreign DNA or plasmids. They are first observed in 1928 by
Frederick Griffith where he proposed that bacteria are capable transferring genetic information
(Havarstein, 2010). These cells such as E. coli are normally made competent by exposure to a rich
calcium environment, where the mixture calcium chloride will be used in. Calcium chloride's
positive charges will cancel out the negative charge of the plasmid and E. coli's cell wall weakening
it, making it easy to pass.
By subjecting these bacteria through sudden temperature changes or heat shock, a difference in
pressure in the outside and the inside of the cell is made, that causes pores, through which the
mobile supercoiled plasmid enters. After normalizing the cell temperature, its walls will heal. When
the E. coli has now taken up the plasmid with GFP, it will be able to grow in agar plates with
ampicillin, an
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35. Pglo Essay
THE EFFECTS OF pGLO
ON E. COLI TRANSFORMATIONS
INTRODUCTION
Green Fluorescent Protein (GFP) is a fluorescent protein found in jellyfish that causes organisms to
fluoresce. This protein was the first fluorescent protein to be discovered and has been highly useful
in a broad range of cell biology disciplines; because of it's highly useful reporter genes and the
ability to use multicolor protein tracking in living cells it has been useful in many scientific
experiments such as E. Coli transformations. There have been many other fluorescent proteins that
have been cloned from a wide variety of marine invertebrates, therefore making GFP not the only
standing fluorescent protein present. (Shaner 2014). The scientific uses of GFP have included ...
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coli bacteria was transformed with a gene that codes for green fluorescent protein. When
constructed, the bacteria grew in the –pGLO LB agar plate, however, was unable to grow in the
other plates. In the –pGLO LB/amp agar plate, there was neither glow or growth, which was
expected, however, the other agar plates were all expected to show growth. Figure 1. picture from
phone with blacklight Figure 2. picture taken with phone without
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37. The Fabrication And Modification Of T Cuas With Cellulose...
2.1 Fabrication and Modification of T–CUAs with Cellulose Acetate (CA) Membrane and
Chitosan–Gold Nanoparticles (CS–GNPs). T–CUAs. Preparation of the T–CUAs follows previously
described procedures.[references] Briefly, the fabrication consist of dropcasting polystyrene spheres
(PSS) of a diameter of 1.54 µm onto a conductive carbon pyrolized photoresist film (PPF) electrode.
[references] In a collaboration with the Stevenson group, we have reported a method for detecting
H2O2 using T–CUAs.[reference] We introduce two modifications to T–CUAs: (1) the addition of a
cellulose acetate (CA) gas permeable membrane selective to NO and (2) the introduction of chitosan
and gold nanoparticles (CS/GNPs) to enhance its catalytic activity, sensitivity, and limit of detection
(LOD) to NO. The schematic of the modified T–CUA is illustrated in Scheme 2. The CA membrane
precursor solution was prepared according to a previous procedure.[reference] The pore size of the
CA membrane (~6 Å) filters out non–gaseous molecules typically secreted by cells (i.e. proteins).
The average membrane thickness is determined to be ~100 nm using atomic force microscopy
(AFM). This is shown in Figure 1.The second modification is introduced to the T–CUAs via one–
step electrodeposition of chitosan gold nanoparticles (CS/GNPs). Figure 2 shows scanning electron
microscopy (SEM) images of an unmodified T–CUA, a modified CS/GNPs T–CUAs. Although, the
size of the GNPs ranges from 5 to 80 nm, there is an
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39. Genetic Transformation And Its Effect On The Green...
The incorporation a plasmid vector, pGLO, into Escherichia coli via transformation is described.
This recombinant plasmid that has been altered contains the ability to fluoresce green under
ultraviolet light because of the Green Fluorescent Protein (GFP). pGLO also encompasses an
antibiotic resistance gene, bla, that allows E. coli to grow in media that contains penicillin derivative
antibiotics such as ampicillin. We conclude that the protein, GFP, is indeed responsible for the green
fluorescence as pGLO contains the gene to encode for it, which is turned on when arabinose is
present. Additionally, when the protein is subjected to denaturation, it decreases the fluorescence
and changes the conformation of the protein structure. ... Show more content on Helpwriting.net ...
Plasmids are the extra–chromosomal DNA in which most bacteria contain in addition to their
bacterial chromosome. Plasmids can be used as vectors to transfer DNA into a host bacterium. A
nonpathogenic strain of Escherichia coli (E. coli) HB101;K–12, is the host bacterium used to take
up the plasmid with the imported gene to be observed. pGLO is a recombinant plasmid that consists
of a gene that codes for Green Fluorescent Protein (GFP) and also contains a gene, beta–lactam
antibiotic (bla), that expresses antibiotic resistance. GFP is a protein that is produced by the jellyfish
Aequoria victoria and has the ability to fluoresce green when exposed to ultraviolet (UV) light. The
gene for GFP can be turned on in the transformed bacterial cell in the presence of the sugar,
arabinose. The bacteria that contain the beta–lactam antibiotic have the ability to create beta–
lactamase, which is an enzyme that hydrolyzes the beta–lactam rings of antibiotics which penicillin
derivatives (among a broad range of others) contain, deeming them incompetent. Beta–lactam
antibiotics inhibit the peptidoglycan layer of the bacterial cell wall and because E.coli is a gram–
negative bacteria, its peptidoglycan layer is easily susceptible to inhibition. pGLO contains
fundamental genes that take part in the replication of the plasmid DNA and the expression of its
traits. bla (indicated by Amp' in Figure A) is a gene that codes for beta–lactamase, which is an
enzyme that functions to
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41. Dna Coding For Ampicillin Resistance And Green Fluorescent...
Abstract This experiment was performed to assess the efficacy of genetic transformations on
bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic
transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and
placing them on various media to observe gene expression via growth and color under UV light. The
transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked
the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of
arabinose, the transformed E. coli glowed green. These results support the previous scientific
understanding of bacterial competency, vectors, and gene expression and support gene
transformations as an effective method to transfer the desirable DNA of one organism into another
organism's DNA. These results can be applied to real world issues such as medical treatments, food
production, and environmental conservation.
Introduction
Genetic engineering is used in health treatments, agricultural applications, and environmental
solutions. Genetic transformations incorporate foreign genetic material into the DNA of a different
organism via a vector, which carries the genetic material. Plasmid DNA is small, round, and
autonomous, due to its origin of replication. In biotechnology, plasmids carry beneficial genes, such
as antibiotic resistance, and also a reporter protein, in this case, Green Fluorescent Protein
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43. Yorktown Case Analysis Essays
Running Head: YORKTOWN TECHNOLOGIES CASE ANALYSIS
Yorktown Technologies Case Analysis
Case Recap
Founded in 2001, Yorktown Technologies, Inc. is a company that specializes in the ornamental fish
industry. The globalization of the ornamental fish industry happened over a half a century ago.
Hundreds of freshwater and saltwater fish can be purchased as pets in virtually any industrialized
nation in the world (Broy, 2011). Yorktown Technologies commercializes a genetically modified fish
called GloFish, which appear to glow in the dark (Mueller, 2010). GloFish are zebrafish that have
been genetically modified with fluorescent colors. They are the first genetically modified animals to
become publicly available as pets ... Show more content on Helpwriting.net ...
The fish are not available in California due to state regulations that ban biotech aquatic organisms
(GloFish, 2011). In addition, Singapore – where the fish was originally engineered – has been
reluctant to approve its sale (Ely, 2004). This places an additional limit on potential marketing and
distribution strategies.
Yorktown Technologies will launch the first commercially available fish that has been genetically
modified. They will be available in the United States in late 2003. They will be available in three
colors: red, green, and orange (GloFish, 2011).
Yorktown Technologies has an exclusive product and market niche. The company has a substantial
number of patents and patent pending applications for GloFish (GloFish, 2011). In addition, the
production, market, and distribution of fluorescent fish are strictly prohibited without the approval
of Yorktown Technologies (GloFish, 2011). This gives the company a monopoly on fluorescent fish
and makes it impossible for other companies to produce, market, and distribute them.
Identifying the Root Problem Components
Yorktown Technologies have several issues that may affect marketing and distribution. The issues
are geographical distribution, production, supply and demand, environmental concerns, and retail
outlets.
First, GloFish are not approved for distribution and sale in California. California is an

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46. Eye Of The Sea Research Paper
Glistening water, beautiful coral composed of your friendly neighbourhood porifera, but what lays
beyond the cliff of coral into deep, dark waters? PhD student, Erika Raymond, and her PhD advisor,
Edith Widder sent the Eye–of–the–Sea to the Bahamas to observe the unknown waters. A variety of
other scientists came up with their own understandings of deep–sea life and how they differ from the
creatures above the waters. One of the characteristics that caught the attention of scientists were the
animals' enlarged eyes that adapted to see bioluminescent materials. Scientists can use this
information to improve their technology to help them find and observe–in the lab or at least
unobtrusively through a camera–deep–sea creatures' natural ways of living. Scientists are interested
in how the different deep–sea species have evolved since the Mesozoic era with very minimal
interactions with humans or man–made objects. However, their habitats are too dangerous for
humans; I suspect there would not have been as much evolution as seen in land animals, considering
the fact that in 2004, Raymond and Widder had spotted a 2 metre squid using Eye–of–the–Sea.
Could there be other organisms that retain their size from the dinosaur ages just because there wasn't
much of a reason for them to evolve? ... Show more content on Helpwriting.net ...
Scientists were dumbfounded when they discovered that deep–sea fish can detect light outside of the
blue–green range (short wavelengths of blue–green light can travel to farther depths underwater,
where you would be able to detect bioluminescence). With further observations, they found that a
species of bottom–dwelling crabs can even detect UV light. In other animals, like the dragonfish,
their 'searchlights' have fluorescent proteins that shift the bluish bioluminescence to the red
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48. Essay On Biofilm Formation
Biofilms are communities of bacterial cells. Bacterial cells become much more antibiotic tolerant by
forming biofilms. Antibiotic resistance causes more than two million infections and 23,000 deaths in
the United States every year. It is really urgent that we need to understand the biofilm formation so
that we can develop novel antibiotics. People have found out that there are some signaling
molecules, which are critical for biofilm formation. However, we do not know how the distributions
of signaling molecules determine individual cell fate during tolerance and persistence development.
Studying the spatial and temporal distributions of signaling molecules in single cell level will help
us understanding the mechanism of biofilm formation. ... Show more content on Helpwriting.net ...
Spinach was discovered using the Systematic Evolution of Ligands by Exponential Enrichment
(SELEX) technique against DFHBI immobilized on agarose resin. People have developed Spinach–
based sensors against adenosine, ADP, S–adenosyl–methionine (SAM), guanine, cyclic–di–GMP (c–
di–GMP) and GTP using existing aptamer sequences. A key challenge for RNA–based sensors is
that they normally function poorly in cells. This is because RNA is easily degraded by nuclease and
also has poor folding in intracellular. Poor folding RNA can be contributed by many elements, such
as the difference folding structures and thermal instability, or rely on ion concentration. Later people
developed 49–nt–long aptamer–Broccoli, which has brighter green fluorescence upon binding
DFHBI. Folding of Broccoli only needs low concentration of magnesium and the Mg2+ presents
low amount in body so Broccoli can fold better than Spinach. Broccoli also has a higher
thermostability than Spinach. Additionally, Broccoli does not require a tRNA scaffold to promote its
folding in vivo. However, under cellular imaging conditions, Spinach and Broccoli show greatly
reduced fluorescence compared with nominal brightness. It has been known that the fluorescence
decrease of Spinach−DFHBI is not due to irreversible photobleaching like GFP. It is caused by
reversible conversion to a non– fluorescent state and the signal can be recovered
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50. Dna Damage, Repair, And Dna Methylation Essay
Introduction:
Modification of damaged DNA seems to be an understudied subject, there is much to understand on
the restoration of DNA damage, repair and DNA methylation. Genomic DNA can be modified by
methylation but much of it is affected on a gene when silenced. When epigenetic modification has
been implicated with cancer and aging it causes DNA methylation to also have an impact on the
double strand of DNA analysis. Modification as such provoke deteriorating changes like aging
found in multicellular organisms and DNA damage may magnify biochemical pathways that
regulate a cells growth or control DNA replication with DNA repair. In the article "DNA Damage,
Homology–Directed Repair, and DNA Methylation" written by Concetta Cuozzo, Antonio
Porcellini, Tiziana Angrisano, et al. they hypothesize how DNA damage and gene silencing may
induce a DNA double–strand break within a genome as well as when DNA methylation is induced
by homologous recombination that it may somewhat mark its reparation through a DNA segment
and protect its cells against any unregulated gene expression that may be followed by DNA damage.
The experiments used to demonstration how gene conversion can modify methylation pattern of
repaired DNA and when that occurs methylation is able to silence the recombined gene. When
exploring the molecular mechanisms that link DNA damage and the silencing gene then there is an
induced double strand break that can be found at a specific location or DNA sequence in where the
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52. How To Write A Pglo Transformation Lab Report
In 1928, Frederick Griffith discovered transformation when observing that living cultures of a non–
pathogenic strain called a Streptococcus pneumonia killed mice after being mixed with a heat–killed
pathogenic strain. Griffith named concluded this as transformation after the non–pathogenic strain
transformed into a pathogenic strain. In 1944, Oswald Avery purified RNA, DNA, and protein from
a virulent strain of bacteria. His experiments not only revealed how virulence the transformation
from S. pneumonia was but, led to the recognition of DNA as a genetic material. The exact mode of
transformation varies from one bacterial species to the next. DNA is transferred between bacteria
using two main methods: transmission and conjunction. In transformation, ... Show more content on
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GFP was first isolated in in the 1970's from the jellyfish Aequorea victoria. Scientist were able to
use the genetic engineering to introduce fluorescent proteins into living organisms once identifying
its specific DNA sequence. Osamu Shimomura, Martin Chalfie and Roger Tsien discovered the
different changes in the patterns of absorption and emission in order to develop a rainbow
fluorescent protein. At this time, the scientists notices that amino acid substitutions in GFP changed
the behavior of the protein responsible for light production or a chromophore. The GFP and its
related fluorescent proteins have become an essential tool in cell and molecular biology. proteins
can be tagged with fluorescent proteins and then expressed in cells using DNA cloning strategies.
Scientist regulate the expression of recombinant proteins using an inducible promoter or a genetic
on / off switch. these sequences allow precise control because expression of the gym only turn on in
the presence of a small molecule. In this experiment, and inducible promoter is used to regulate GFP
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54. Lab Report Green Fluorescent Protein
Abstract section
Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with
high fluoresce and absorbance. The purpose this experiments is to purify and express a His2–tagged
recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA–GFPUV > through
a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuv
gene (UV–optimized GFP) was over expressed in the E. Coil strain BL21 (DE3) (pLysS) as an n–
terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity
chromatography to obtain purification of the crude extract. Then observe under the long wavelength
UV light, the activity of the rGFP in the column fraction. Bradford assay was performed to obtain
the total protein amount. When calculating the ... Show more content on Helpwriting.net ...
Below is the map of pRESTA–GFPuv in Figure 1. This map shows us that IPTG was induced first in
the system, making the lac repressor inhibited, this then expressed DNA T7 polymerase. This makes
the T7 promoter to create the protein of rGFP is transcription. G0 means time equals zero.
FIGURE 1: An expression plasmid map. rGFP contains a total of 289 amino acids from the GFPuv
DNA sequence shown in Figure 2. There is 238 amino acids in the rGFP and 38 amino acids in
His6tag/Xpress epitope. His 6 tag comes from the N terminus.
FIGURE 3: A schematic diagram of the rGFP protein.
In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs
from the Ni+2 Agarose column. E3 had the highest elution fraction with 8554 RFUs. In the
Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was
about 1.28E5 RFUs/mg. Overall, in the Figure 3, the E1–E6 had most of the rGFPs due to being
bonded in Ni+2. W1–W6 rGFP is much smaller than the E1–E6 amounts.
FIGURE 3: The combined elution
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56. Gene Cloning And Bacterial Transformation
Introduction: In this lab, gene cloning and bacterial transformation was conducted. Bacteria are
single celled microorganisms. They are simple in structure and have no nucleus with linear
chromosomes. Bacteria are essential to life, and reproduction of other bacteria which helps play a
role in decomposition of organic matter and the cycling of elements such as nitrogen and carbon,
which are elements necessary to live. Due to this, plants and animals can use nitrogen to create
nucleic acids along with amino acids, which are the essential building blocks of DNA. They also
help break down food in the digestive system, which allows us to obtain the nutrients we need, and
bacteria helps develop our immune system to fight other illnesses that ... Show more content on
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The bacterium being observed was E. coli, which codes for green fluorescent protein. E. coli is a
bacterium that normally lives in the intestines of humans and animals alike, and can cause infection.
One hopes to observe a glow from a green fluorescent protein (GFP) which is typically made by a
jellyfish. This will be made possible by the additional presence of arabinose, which acts as the
inducing agent. The inducing agent influences the transcription of the GFP and activates the
transcriptional promoters. A promoter allows a foreign gene to fall into place much easier resulting
in gene expression. It is expected that the combination of the plasmid, with the Lysogeny Broth (LB)
and the arabinose (ARB) will transform the bacteria, and allow for growth of the bacteria in the
presence of the antibiotic ampicillin (AMP) along with an observable glow under ultraviolet (UV)
light.
Hypothesis:
The Agar plate with the LB/AMP/ARB in the +pGLO will be the only plate to display growth and a
glow when compared to the other agar plates.
Materials:
Water bath set at 42 degrees Celsius
Pipette tips
Micropipetter (P20)
50 mM CaCl2 solution
pGLO plasmid
Sterile loop
Competent E. coli HB101
37–degree incubator
Sterile LB agar plates (LB, LB/amp, and LB/amp/ara)
Sterile transfer pipette

57. Methods:
1. After labeling each of the tubes, 250 mL of CaCl2 was added to all the tubes and
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59. Pglo Lab
Abigail Soberano Zapata
Life 102 Lab L13
Lab Report
The effects of green fluorescent protein on E. coli bacteria
Introduction:
Genetic transformation occurs when genes are inserted into another gene to change the organism's
trait (Weedman2016). In this experiment, we proceeded to transform the E. coli bacteria with a gene
that contained green fluorescent protein. The green fluorescent protein is used in experiments
because it beams a green color under a UV light (Chalfie2008). Typically, it is used to mark the
expression of genes, which is why it serves as the symbol for all gene expressions (Tsien1998). In
the experiment, we will be using pGLO as the organisms that will transmit the disease, otherwise
known as a vector. The pGLO in the experiment ... Show more content on Helpwriting.net ...
"Green Fluorescent Protein." Photochemistry and Photobiology 62.4 (2008): 651–56. Web. 7 Nov.
2016.
Froger, Alexandrine, and James E. Hall. "Transformation of Plasmid DNA into E. Coli Using the
Heat Shock Method." Journal of Visualized Experiments ser. 2007.253 (6) Web. 22 Nov. 2016.
Hinton, Page. "The Effect of the Insertion of the pGLO Plasmid on E. coli's" Prezi.com. 10
December 2013Web. 7 Nov. 2016
Tsien, Roger Y. "THE GREEN FLUORESCENT PROTEIN." Annual Review of Biochemistry 67
(1998). Howard Hughes Medical Institute. Web. 7 Nov. 2016.
Weedman Donna. 2016.Life102.Attributesoflivingsystems.
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61. Green Fluorescent Protein Lab Essay
Title: Purification of Green Fluorescent Protein
Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria.
Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel
electrophoresis is used to check and analyze the pure protein. Research scientists use Green
Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and
multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC.
Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).
Purpose: To illustrate the process of transformation and perform it using Green Fluorescent Protein.
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Invert.
16. Microcentrifuge for 1 minute. Use a micropipette to transfer the supernatant containing the
recombinant GFP to a new 1.5mL Eppendorf tube.
17. Observe the GFP under ultraviolet light.
Data: (Laboratory Procedure for pGREEN)
1./2./3. LB–plasmid: LB+plasmid: LB/Amp+plasmid: LB/Amp–plasmid
Prediction: average growth average growth growth no growth
Reason: control control it has LB/Amp and plasmid no plasmid
Result: lawn lawn 17 colonies no growth
4.
LB+plasmid (Pos Control) lawn LB–plasmid (Pos Control) lawn
LB/Amp+plasmid (Exper) 17 colonies LB/Amp–plasmid (Neg Control) no growth
5.
a. LB+plasmid and LB–plasmid: Both of these plates had a lawn of bacteria. This proves that the
bacteria are capable of growing on the agar and that there was nothing preventing growth beside the
ampicillin.
b. LB/Amp–plasmid and LB–plasmid: The LB/Amp–plasmid had no growth compared to the LB–
plasmid which had a lawn. This proves that transformation cannot take place without the plasmid, or
DNA.
c. LB/Amp+plasmid and LB/Amp–plasmid: The LB/Amp–plasmid had no growth, but the
LB/Amp+plasmid had growth. This shows that the bacteria was transformed and

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64. The Bacteria Escherichia Coli ( E. Coli )
Question 1
In the past scientist have used the bacteria Escherichia coli (E. coli) in Synthetic Biology to allow
the bacterial species to recognise and respond to light frequencies providing greater knowledge of
complex biological systems (Levskaya et al. 2005). E. coli is a suitable chassis to express Green
Fluorescent Protein (GFP) as the bacteria's plasmid helps to establish GFP (Chavshin et al. 2013).
The bacterial plasmid is a maltose–binding protein that helps E. coli and GFP fuse together and
allows for detection of gene expression and controlling internal environments (Feilmeier et al.
2000). As GFP is a useful biological indicator due to its fluorescent structure (Mazzola et al. 2005)
and through the ability of E. coli responding to light, GFP is easily expressed through the process of
light stimulation (Lee, J et al. 2013). Through the unique structure and functions found within E.
coli, it allows for successful expression of GFP making E. coli a suitable base organism.
Question 2
Jellyfish (Aequorea victoria) naturally produce GFP (Zupen et al. 2004) allowing for extraction of
the organisms genes and protein to engineer a bacteria that can produce GFP. Scientists are able to
blind a bacterial species for example E. coli with GFP by cloning the genes found within jellyfish
that produce Green Florescent Protein and insert it into a chosen bacterial species (Zupen et al.
2004). Through the expression of the GFP gene in other organisms, synthetic biology can be
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66. Pglo Transformation Essay
Connor Lauffenburger
3/17/13
pGlo Transformation Lab Report
I Introduction
The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a
plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that
confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate
independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found
in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one
organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were
transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies ...
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Next, the transformation efficiency was calculated by dividing the total amount of DNA on the agar
plate in μl by the number of cells growing on the +pGLO LB/amp/ara plate.
During the purification section of this lab, the LB/amp/ara agar plate was examined for well–
isolated green colonies and the LB/amp plate was observed for white colonies with space between
each other. These colonies were circled on the outside of the plates using a marker. Next, two 15
milliliter culture tubes containing 2 milliliters of nutrient growth media were obtained and labeled
"+" and "–". Using a new inoculation tube, the circled colonies from each plate were scooped out
and immersed in their respective culture tubes. Once the bacteria was mixed into the solution, the
tubes were sealed and placed horizontally into the 32⁰ incubator for 24 hours.
After labeling a new microtube with a marker, the culture tubes were removed from the incubator
and observed under the ultraviolet lamp. Using a sterile pipette, the contents of the (+) culture tube
were transferred to the 2 milliliter mictrotube and the (–) culture tube was disposed of. The (+) tube
was then spun in the centrifuge at maximum speed for 5 minutes. Once it was removed from the
centrifuge, a noticeable bacterial pellet had formed at the bottom of the tube and the remaining
liquid was disposed of. After the
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68. Green Fluorescent Protein Chromatography Lab Report
Purpose: Green fluorescent protein (gfp) and blue fluorescent protein (bfp) extracted from bacteria
were separated by chromatography by means of a molecular sieve matrix. This process utilized the
proteins size and shape and was able to separate it from the other molecules and be collected in test
tubes. The tubes containing the desired protein will glow under U.V. light. By running an
electrophoresis gel with these proteins, one can compare the protein bands and be able to determine
the approximate molecular weight of the proteins. In this experiment, students learned about the
methods and procedures to partially purify gfp and bfp. After purifying the proteins, the students
will be able to compare the molecular weights of the samples by running a denatured SDS
polyacrylamide gel (Purification & Size Determination of GFP & BFP, EDVOTEK). This will show
students which proteins different from one another and how denaturing can affect the proteins.
Background: ... Show more content on Helpwriting.net ...
When energy is transferred to these proteins it causes them to emit light. They will also glow when
under U.V. light (Purification & Size Determination of GFP & BFP, EDVOTEK). The proteins are
extracted by using size–exclusion chromatography. This is a method where a solution is prepared
and separates molecules based on their size. The fluorescent properties of these proteins make them
heavily sought after when making chimeric proteins. Scientists will fuse bfp or gfp to another
protein to use it as a biomarker. These fusions will typically not affect the biological activity of the
chimeric protein (Purification & Size Determination of GFP & BFP, EDVOTEK). This makes
fluorescent proteins a tool that allows science to locate specific proteins within a cell and what their
purpose
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70. Prevagen Case Study
so I was curious about this pill that claimed it could support memory and improve brain function the
pill is called "Prevagen" and makes many claims about what it can do. this was one of the articals I
read about this so called "magical pill." " The biggest claim is that Prevagen improves memory,*
something pretty much everyone would like. The website also includes the more specific claim that
?Prevagen can improve memory within 90 days.*? The package adds that Prevagen ?supports
healthy brain function,* [a] sharper mind,* and clearer thinking.*? "How can they make these
claims if they aren?t true? Simple: every claim has a little asterisk (*) next to it. If you scroll all the
way to the bottom of the Prevagen webpage, you?ll find what that
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72. Brainbow Essay
Brainbow is a powerful tool used to visualize microscopic biological systems like cells, tissues and
organelles for the purpose of studies. During histogenesis of the nervous system, neurons beget
neurons as well as glia which proliferate, divide, migrate and differentiate. However due to their
microscopic nature, similarity as well as proximity to each other, tracing their lineages, path of
migration and final destination becomes complex and makes their study almost impossible. This
puzzle was resolved after the discovery of color (green fluorescent) expressing genes Aequorea
victoria (Shimomura et al.1962), which is capable of visualizing cells, tissues organelles and their
interactions (pioneered by Chalfie et al. 1994) based on distinctive ... Show more content on
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Based on this analogy, multiple fluorescent genes can be genetically engineered to express varying
fluorescent proteins making each closely related neuron as unique as possible. One efficient
technique utilized in making a brainbow mouse is the Cre/lox–based recombination technique which
yields pattern of protein expression of different colors. It has alternative means like recombinase–
mediated DNA excision–based/ inversion–based both of which combine to produce about four
different combinations of genes compared to other techniques. In the recombinase–mediated DNA
excision, three unique FPs are arranged in order along with two–pairs of Cre recombinase
recognition site (Lox site) in between two of the FPs in the transgene. Identical pairs of the two
recognition sites can only be detected by Cre. Prior to recombination, the first color (default) is
expressed. After recombination, one of the FPs is expressed from what has now become a "cassette".
In the Invertion–based brainbow however, homogeneous pair of recognition sites are placed to face
each other. The interspaced DNA is inverted by Cre. With two FPs aligned anteriorly, the inversion
causes one of the FPs to be expressed (Livet et al.
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74. Human Ipsc Line, 201b7 Clone Essay
1. The source of the iPSC's in this case study are human iPSC line "201B7 clone". They are dermal
fibroblast tissues that in previous studies have been shown to have low tumorigenicity (meaning
they have a low ability to produce tumors) after being used in transplantation therapy.
2. The iPSC's were different from the host cells because a piggyBac vector was introduced into the
hiPSC. A piggyBac vector is a genetic element that literally does what it sounds like, gives a
piggyback between vectors and chromosomes, and cuts genes, carries them, and pastes them into a
new position. This specific piggyBac vector used to distinguish the host cells from the iPSC's stably
expressed GFP (green fluorescent protein) gene under a ubiquitous promoter. The promoter was EF1
(elongation factor used to push gene expression in vitro and in vivo). The authors knew their
transplantations of iPSC's were successful because once the cells were introduced they observed
continuous GFP fluorescence. The GFP fluorescence was observed even after differentiation of
neural–linage.
3. iPSC's were differentiated into neural stem cells by using SMAD–pathway inhibition. Green
fluorescent proteins were labeled iPSC, and then a "serum– free floating culture of embryoid body–
like aggregates method" was combined with the SMAD– pathway. These methods stimulated the
LIF/BMP signaling. Leukemia inhibitory factor and bone morphogenic protein both promote neural
cell differentiation into GFAP (Glial
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76. Two Control Plates Lab Report
The two control plates LB AMP–PGLO and LB AMP+PGLO (refer to Figure 3.0 and Figure 4.0)
include a nutrient causing growth and the antibiotic, ampicillin, with or without the plasmid DNA.
The purpose of the two is to provide a determination of the LB AMP ARA+PGLO and whether or
not, the plasmid has an effect with the sugar. In addition, the two controls show that plasmid, pGLO,
can affect the growth of bacteria with the consideration of ampicillin. As well, the two control plates
prove that the results of the LB AMP ARA+PGLO and the LB–PGLO plate can be reliable knowing
that they have been under the same conditions of the incubator as the control plates. Green
Fluorescent Protein acts as the biological molecule for the fluorescence. In fact, ... Show more
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Firstly, the lab was done within a period of 75 minutes. Time efficiency was a factor that could have
been more accurate when creating competent cells. Competent cells are able to take in extra foreign
DNA. The procedure involved timing for series of events. Events such as: both tubes on the ice for
10 minutes, both tubes in the bath for 50 seconds, both tubes on ice for 2 minutes and both tubes at
room temperature for 10 minutes. All of these events could have been done with greater precision.
Some of the tubes may have been exposed to their given environment over or under the necessary
time frame. A source of error is that the ice blocks were not consistent. The procedure asked for
crushed ice and ice cubes were used in the experiment. This could have had an effect on the
temperature needed for the creation of competent cells. On the other hand, an improvement that
could be made is pipetting. 0.25mL of CaCl2 solution was put into both tubes using an open pipette.
But, it was possible for a mistake to occur with the chance of air bubbles or imprecision to the
required amount. This would affect the amount of the CaCl2 solution that was needed by under or
over pipetting into each of the two test tubes. Lastly, a source of error may be how spread the
substances were amongst the plate. The greater the distance spread around the surface of the plate,
the possibility of more growth and less bacterial lawns formed on the four
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78. Cameleon Analysis
3.1.1 FRET–based Ca2+ indicators
Cameleons, the first generation of GECIs [161], are chimeric proteins that, in their original iteration,
were composed of a blue or cyan FP as a FRET donor and a green or yellow FP as a FRET acceptor.
The two FPs are connected by CaM and its binding partner M13 (the CaM–binding domain of
myosin light chain kinase) (Figure 6A). A similar design with only the CaM–binding domain was
also reported in 1997 [162]. When the CaM domain of a cameleon binds to Ca2+, it changes its
conformation and sequentially binds to M13, which results in a more compact conformation that
decreases the distance between the FRET donor and acceptor. This decreased distance causes an
increase in FRET efficiency that manifests itself as ... Show more content on Helpwriting.net ...
The presence of endogenous interaction partners may interfere with GECI function and lead to
reduced or diminished dynamic range [156]. In 2004, Griesbeck and coworkers engineered a new
type of FRET–based GECI, called TN–L15, by replacing calmodulin with troponin C (TnC) which
interacts with fewer endogenous proteins [167]. In 2006, Mank et al. reported further improvements
in the dynamic range and kinetics of TnC–based indicators by engineering the magnesium– and
calcium–binding properties within the C–terminal lobe. The best variant to arise from that work,
TN–XL, exhibits a highly reproducible fluorescence signal with fast rise and decay times, as
demonstrated in the presynaptic motor neurons of transgenic flies [168]. By deletion of low affinity
N–terminal lobes, the same group obtained a further improved version of TnC–based GECIs,
designated TN–XXL, with higher affinity to Ca2+. They demonstrated the usefulness of TN–XXL
for the chronic imaging in mouse brain in vivo [163]. Very recently, Thestrup et al. developed a new
FRET–based indicator 'Twitch', based on Opsanus troponin C, with reduced number of Ca2+–
binding sites compared to TN–XXL [149]. They optimized the indicator by combining a large
functional screen in bacteria with a secondary screen in rat hippocampal
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80. Photosynthesis Lab Report
1. The technique that allows scientists to follow with their own eyes the dynamic movements of
specific proteins as they occur within the living cell is the green fluorescent protein. This is a protein
that is extracted from only a few jellyfish. This diffuses a green fluorescent colored light. The green
fluorescent protein can be blended to the protein and can work normally and so does the protein
itself that it is binded to. The protein is not affected and can be moved and transported throughout
the cell and can then be seen.
2. The early endosomes progress gradually. The early endosomes transform into the late endosomes
because of the lowered levels of ph, when Rab proteins are traded, and the earlier endosomes
internal structure has a crucial ... Show more content on Helpwriting.net ...
The HIV virus must be have reverse transcriptase in order to duplicate the virus. AZT restricts the
reverse effect of the transcriptase and it could potentially effect the transposition. This would mean
that it must be reversed transferred into only completed DNA. That DNA would mean it must be
double stranded and directly implanted into the target site. By preventing the reverse transcription
with the AZT drug should stop the process at an early stage.
4. In order the amino acid sequence to be maintained, the function of the molecule must also be
preserved and maintained. The changes are not allowed to be selected against it. But, if the amino
acid sequence were to be changed due to mutation, then the performance of the molecule could
change relatively badly. If this were to happen, then this would show that the changes would be
selected against it and disappear.
5. The mRNA inconsistency permits an accelerated response to the environment changing if the
synthesis of a new mRNA is permitted if older, not needed ones, can fade or vanish because they are
inconsistent. Cells cannot create copies of proteins on their own at a fast pace and therefore would
get nowhere if the proteins were created off of the DNA template strand precisely. mRNA admits
one of the DNA molecules to supply the template strand but in the shape of several mRNA
molecules. Therefore, an abundant amount of polypeptide chains are created and allowing the cell to
manufacture an intensified amount of
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