Kaushik RameshFinal Paper

pAcGFP1-Mem and GFP in cellular Membrane Experiments

Abstract

The GFP gene is a useful tool in cellular biology due to its green fluorescence.

By joining GFP gene to the gene of the protein of interest, researchers can see where

proteins are made, and where they can go. The same logic is applied to the vector

used in this experiment, pAcGFP1-Mem, which encodes for the Neuromodulin

protein. This protein localizes to the cellular membrane, which allows the

membrane to be observed through the GFP’s green fluorescence. In this experiment,

we transform the genome of the cell to hold this pAcGFP1-Mem vector. Through

various tests using Gel-electrophoresis and the selectable marker, we confirm the

successful transformation of the genome. Furthermore, we observe the green

fluorescence of the GFP ourselves through the use of a confocal microscope. Our

tests indicate that the transformation was successful and the confocal microscope

clearly showed that the membrane was highlighted by the GFP. The results of our

experiment indicate that transformation through the pAcGFP1-Mem vector can be a

fast, simple, and reliable method in experiments researching the cellular membrane.

Introduction

Being able to observe and analyze the membrane is an important step in

many different experiments. For example, it would be useful to examine the

mitochondria when testing for what effects osmosis has on the cell volume (1).

Experiments like these attempt to examine the membrane using imaging techniques

such as atomic force microscopy or more invasive techniques involving

fluorescence. But techniques involving fluorescence can cause issues because of the

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difficulty in labeling cells with fluorescence type proteins; the problems with using

fluorescence proteins include getting the genome to accept the gene and being able

to express the fluorescence. The pAcGFP1-Mem vector alleviates many of these

problems due to the ability to integrate the green fluorescence protein gene directly

into the desired genome.

Techniques involving the green fluorescent protein (GFP) are alluring

because the GFP gene can be integrated into many different organisms using vectors

and further maintained with breeding. GFP can be linked to cellular control factors

like promoters in order to study cell growth. For example, in a study by Kawaja, The

GFP was linked to promoters in order to study what triggers cells to grow in nerves;

when the nerves begin to grow, the cells start growing green (2). GFP can also be

used with antibodies. Like in the experiment by Leveau, GFP can be used and

measured with antibodies in order to determine if a culture of bacteria are growing

(3). Other uses for GFP include finding out if a protein binds to another cellular

protein, if a stem cell transplantation succeeded, and if cancer has reached its

metastasis point. Even if the GFP protein has folded incorrectly and is being

expressed in a non- fluorescence form, antibodies can bind to the malfunctioned

GFP and determine in what stage the failure occurred—for example, was there a

problem with, protein folding, protein synthesis, or gene expression.

In the experiments described here, the vector— pAcGFP1-Mem—was used to

provide a simple way to deliver the protein gene into the genome. Once this is done,

the GFP can be used in experiments in any of the ways previously mentioned. In this

report, we will provide a detailed analysis of the success of the pAcGFP1-Mem using

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tools like the confocal microscope and gel electrophoresis. This is to determine if

the pAcGFP1-Mem can deliver the GAP gene successfully into the genome in order to

conduct experiments like those mentioned above. If we find that the pAcGFP1-Mem

is very efficient at integrating the GAP gene into the genome and that the GFP is

being delivered to the cellular membrane properly, it can be utilized as a simple and

fast tool in any experiments involving membranes.

In conclusion, I believe that these experiments will show that the pAcGFP1-

Mem vector will be an indispensable tool in researching the cellular membrane

because of its simplicity and efficiency in transforming the target’s genome.

Hypothesis: pAcGFP1-Mem vector successfully integrates the GFP gene into the

genome. Also, GAP-43 localizes the GFP into the cellular membrane. The green

fluorescence on the cellular membrane is clearly visible and distinguishable from

the rest of the membranes on the cell.

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Methods

Plasmid

The plasmid contains a CMV promoter from 1 – 589. Three origins of replication

which are f1, SV40, and pUC. The f1 origin of replication allows for single stranded

replication and packaging. The SV40 is a mammalian origin of replication by NIH-

3T3 cells upon transfection. The pUC is the bacterial origin of replication and is able

to make a high copy number of around 500-700. The selectable markers are the

kanamycin and neomycin resistance genes and they are located at 2683 – 3211. The

neomycin resistance gene selects for prokaryotic cells while the kanamycin is made

for eukaryotes. There are also three restriction enzymes that are coded from this

vector: BamH 1 which is at the 661 location, Age 1 which is on 667, and Not 1 at

1456. They are all type 2 restricton enzymes which means that they wont be

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Figure 1: pAcGFP1-Mem Vector

effected by DNA methylase. They all also have an incubation temperature from

around 30-37°C and two of them have an inactivation temperature of 65°C; only

BamH 1 doesn’t have an inactivation temperature. Finally, there is a AcGFP1 fusion

gene from 679 – 1452.

Cell Culture

Four cell cultures were made. The setup:1 – B-Gal Experimental 2 – B-Gal Control 3 – Membrane Vector Experimental 4 – Membrane Vector Control

The B-Gal E.coli was treated with Ampicilin while the Membrane culture was treated

with Kanamycin.

Gel Electrophoresis

The gel Electrophoresis was done to ensure that we had the succesful DNA

recovery in prokaryotes. The Gel and components can be seen in figure 3 and 4.

Microscope

To confirm the success of the GFP gene into the genome, we used a confocal

microscope to anaylze the green fluroscent within the cells.

Western Blotting and PCR preparation

By using Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction

reagents, we were able to separate cytoplasmic extracts from the cultured cells.

Adherent cells were harvested with trypsin and then centrifuged for 5 minutes at

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500 x g. Suspension cells were centrifuged right away. Next, the cells were washed

with PBS and centrifuged once again. Finally, beofre the reagents were added, the

supernatant was discarded, leaving only the dry dell pellet. The CER I and CER II

reagents cause cell membrane disruption which, in turn, releases the cytoplasmic

contents. The reagents must be kept in ice cold before the experiment and the

sample must be vortexed for 15 seconds and incubated for 10 minutes after adding

CER I and 5 seconds of veortexing with 1 minute of incubating after adding CER II.

Next, the NER reagent allows the proteins to be extracted after centrifugation. The

strength of this reagent is that it yields products with less than 10% contamination

between nuclear and cytoplasmic fractions. This amount of purity is useful for more

steps with these components. An insolubule pellet will form by vortexing the tube

for 5 seconds and centrifuging for another 5 minutes. Suspend this pellet in ice-cold

NER. Continue vortexing the sample every 15 seconds for 10 minutes, for a total of

40 minutes while placing the sample on ice during the breaks. Finally, centrifuge at

maximum speed at around 16,000 x g for 10 minutes and the nuclear extract will be

ready for the next step.

RT-PCR

The tubes for the RT-PCR lanes were made as follows.

PCR Tube Content1 RNA + GFP Primer2 RNA + Protein Primer3 RNA + No Primers4 RNA + GFP, no RT enzyme5 RNA + Protein, no RT enzyme6 No RNA + GFP Primer7 No RNA + Protein Primer

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Figure 3: Table of contents for each PCR tube

Results

Cell Culture

The expected vector transformation color was white. Again, our setup was:

1 – B-Gal Experimental 2 – B-Gal Control 3 – Membrane Vector Experimental 4 – Membrane Vector Control

Cultures 1 and 3 ended up being a darkish blue color. Cultures 2 and 4 didn’t seem

to survive. This proves that the transformation of the vector to the bacteria was

successful.

Gel Electrophoresis

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Figure 3 and 4: Gel Electrophoresis and table of lanes

Lane Content1 Ladder2 Cellular Vector 13 Cellular Vector 24 Cellular Vector 35 Cellular Vector 46 Prokaryotic

Vector 17 Prokaryotic

Vector 2

Lanes 8-12 are empty.

The expected size of the B-Gal was 6820 base pairs while the membrane was 5800

base pairs. This seems to correlate with our data because the B-Gal lanes seem to be

slightly thicker. Our data in the Gel Electrophoresis also seems to correlate with the

Spectrometer Data in the next section. The thicker lanes have the higher

concentration and absorbance compared to the other lanes. For example, lane 4 had

the highest values with a concentration of 401.8 n/uL and an absorbance of 8.035

nm; its respective band, at lane 5, also seems to be the thickest.

Spectrometer Data

Sample Concentrationn/uL

Absorbance260 nm

260/280 nm ratio

Membrane 1 81.8 1.636 1.81Membrane 2 323.8 6.475 1.87Membrane 3 63.5 1.269 1.84Membrane 4 401.8 8.035 1.81Amp 1 4.6 4.3 1.84Amp 2 318.7 6.373 1.84

Confocal Microscope Visual Analysis

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Figure 6: Confocal Microscope Image

Figure 5: Table of Spectrometer Data

Due to the flexibility of the confocal microscope, we can observe many different

layers of our cell. An interesting fact we found was that we see more fluorescence at

the top of the cells. This is due to the cells being polarized. Also, not all of the cells

had strong green fluorescence. This is due to our transient system, causing our

daughter cells to have less of the vector. Our GFP also partially labels other

membranes within the cell, such as the mitochondrial membrane. This can be seen

because some portions of the cell seem to have a brighter highlight than others;

these brighter portions indicate the GFP highlighting some of the intracellular

membranes.

RT-PCR Results

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Figure 6: Confocal Microscope Image

Figure 7: RT-PCR imageFigure 8: Table of Lanes on RT-PCR Gel

1 Ladder Lane2 RNA + GFP Primer

3 RNA + Protein Primer

4 RNA + No Primers

5 RNA + GFP, no RT enzyme

6 RNA + Protein, no RT enzyme

7 No RNA + GFP Primer

8 No RNA + Protein Primer

Lanes 9-12 are empty

Our RT-PCR experiment was not very successful, as the lanes seemed to be

extremely light. Perhaps the solutions used to fill the protein and GFP primer lanes

were too diluted. If they were washed down with too much water, it would cause

some of the primers to not function and thus become much lighter on the gel.

Moreover, lanes 3 and 4 are expected to be empty because 3 contains no GFP while

4 contains no primers at all. Also, both lane 7 and 8 had no RNA so they were

supposed to be empty as well. As seen on the picture though, both lanes seem to

have a faint band.

Discussion

GFP can be very useful in experiments revolving around the cell membrane

due to its fluorescence ability. As mentioned before, one way to utilize GFP is to

determine the effects of osmosis on the cellular membrane (1). The vector used in

this experiment—pAcGFP1-Mem—is incredibly useful in transferring the genetic

material for GFP to the desired organism. The ability of the pAcGFP1-Mem vector to

transform into the genome was tested in these experiments; after the

transformation was complete, a gel-electrophoresis was done in order to ensure the

DNA recovery in prokaryotes. Furthermore, an RT-PCR was also conducted in order

to confirm the DNA recovery by the PCR primers. The actual visual analysis itself

was done through the use of the confocal microscope. A western blot was planned

to confirm recovery of the protein of interest, but ultimately failed due to faulty gels.

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pAcGFP1-Mem in particular codes for the Neuromodulin protein (GAP-43).

GAP-43 is transported to the membrane protein through the use of its sequence on

the N-terminal region. This sequence at the N-terminal region helps shuttle the

GAP-43 protein to the membrane. Naturally, GAP-43 has a role in making and

maintains connections between neuron cells. In our pAcGFP1-Mem vector, we use it

as a messenger of GFP to the cellular membrane. In an experiment headed by

Udvadia, researchers used GFP and GAP-43 in a similar way us in researching

differences in axon growth between CNS development and regeneration (4).

The most relevant data for the usefulness of the pAcGFP1-Mem in

researching for changes in the cell membrane is the images from the confocal

microscope. The images are clear proof that the pAcGFP1-Mem was integrated into

the genome because we can observe the green highlighting of the GFP. The confocal

images show that the top layer of the cell seems to have more fluorescence than the

middle or the last few layers. This might be due to the polarity of the cell and

because the GFP is more concentrated in the first few layers. In addition, the higher

amount of fluorescence near the center of the cell comes from intracellular

membranes that also have some GFP. Experiments that only need data on the

cellular membrane might have difficulty due to the fact that some intracellular

membrane might also receive some green fluorescence. While this might make GFP,

and thus the pAcGFP1-Mem, unreliable in some cases, it may also lead to interesting

results. In her experiment, Castaneda took advantage of GFP’s tendency to highlight

some organelle membrane to test the differential expression of the CB2 protein in

extracellular membranes vs intracellular membrane (5). Thanks to this supposed

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downside of GFP, she was able to find the human PBL cells express the CB2 protein

at the intracellular membrane while B cells express them at the extracellular

membrane.

In another experiment, Porcelli attempted to detect differences in pH across

the outer mitochondrial membrane (6). Unlike all the previous experiments in

using GFP to study the mitochondrial membrane, these experimenters used GFP to

exclusively examine the mitochondrial membrane and completely circumvented the

cellular membrane. The experiment concluded by determining that the pH of the

mitochondrial matrix was higher than the inter-membrane space.

As we can see from the confocal data, the pAcGFP1-Mem can efficiently

highlight extracellular membranes. But does this mean that the pAcGFP1-Mem

vector is a good tool to be used in experiments that need to extensively analyze

cellular membranes? There seems to be two prime weaknesses from the data. First,

the GFP can be localized unevenly due to external factors like polarization. Second,

GFP can also highlight some intracellular membranes. As we can see in experiments

from Castaneda and Porcelli, GFP localizing in some intracellular membranes can be

taken advantage of in order to gain more results about the cell as a whole. Thus, this

“weakness” of the GAP-43 of the pAcGFP1-Mem localizing the GFP into some

intracellular membranes can also be seen as a strength. The other weakness of the

GFP randomly localizing unevenly along the membrane can be simply overcome

through multiple experiments. While this might end up costing more money and

taking more time, it can eliminate any random factors the GFP might have in

localizing along the membrane. In conclusion, pAcGFP1-Mem is a very useful vector

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in studying changes on the cellular membrane. In addition, it also allows

researchers to investigate any changes within the intracellular membrane as well.

Thus, researchers can use this vector as a simple and fast method in reliably

studying both the extracellular and intracellular membranes.

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References

1. Becker,D and Hahn,J. (2007), “Functional interaction of the cation channel transient receptor potential vanilloid 4 (TRPV4) and actin in volume regulation”, http://www.sciencedirect.com/science/article/pii/S0171933508001441

2. Kawaja, Michael D. et al. “Nerve Growth Factor Promoter Activity Revealed in Mice Expressing Enhanced Green Fluorescent Protein.” The Journal of comparative neurology 519.13 (2011): 2522–2545. PMC. Web. 6 May 2016.http://www.ncbi.nlm.nih.gov/pubmed/21456011

3. Leveau, Johan H. J., and Steven E. Lindow. “Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria.” Journal of Bacteriology 183.23 (2001): 6752–6762. PMC. Web. 6 May 2016. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95514/

4. A.J. Udvadia, and R.W. Koster, (2001), “GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration” http://dev.biologists.org/content/128/7/1175.article-info

5. Castaneda, Julie T. et al. “Differential Expression of Intracellular and Extracellular CB2 Cannabinoid Receptor Protein by Human Peripheral Blood Leukocytes.” Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 8.1 (2013): 323–332. PMC. Web. 6 May 2016.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587044/

6. Porcelli, A.M. and Ghelli, A. (2004), “pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant”http://www.sciencedirect.com/science/article/pii/S0006291X04026890

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