Charles Bluford 1

Cancer Gene Fusion

Charles Bluford

Notre Dame College

Dr. Johnson (2016)

Charles Bluford 2

Abstract

The purpose of the experiment was to create the mutant cancer gene that develops

through the combination of Jaz-f1 and Su(z)-12 by using E.coli bacteria. Through creating the

cancer gene, it is possible to then inject that gene into an embryonic Drosophila melanogaster,

create a stable population of cancerous fruit flies, and then manipulate the cancer gene to observe

the various effects that it has on the flies. The variables of the experiment were making sure that

the ecoli had taken up the Jaz-f1 and the Su(z)-12 and obtaining the proper DNA annealing

temperature for the Taq PCR. At the end of the experiment, the 2 pieces of DNA were able to

anneal to the 3 prime and 5 prime regions and allowed for the formation of the fusion cancer

gene. The next step in this process would be to then introduce this gene into a new generation of

flies. This experiment has just been proven last month on November 8 using small cell lung

cancer fusion with mouth genes. Their results showed that the mutant could be established and

show positive effects on a mouse using the Jaz-f1-Su(z)12 fusion protein to disrupt the PRC2

complex. Their findings will allow researchers to now study the stages of human endometrial

stromal tumorogenesis at the chromosomal level and make progress for creating stronger

treatments to combat this disease.

Methodology

The first step of the experiment was to culture 2 sets of E.coli bacteria: One with the Jaz-

f1 and the other with the Su(z)-12. The intermediate steps included thawing fresh E.coli bacteria

for the experiment. Next, 2 µL of plasmid containing the Jaz-f1 and Su(z)-12 was added to the

respective cultures. This was done so that the E.coli has a way to absorb new DNA in the

immediate area. There were 6 Jaz-f1s and 10 Su(z)-12s. The 2 vials of E.coli were then put on

ice to incubate for 20 minutes. After which, they were heat shocked at 42 for 2 minutes. This ℃

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opens up the pores of the E.coli and allows for the absorption of the plasmids containing Jaz-fl

and Su(z)-12. Next, they were put on ice for 5 minutes to allow for the closing of those pores.

Lastly, the 2 specimens of E.coli where then given 500 µL of LB ampicillin and was incubated at

37 for 1 hour, and then re incubated on a plate at 37 overnight. If E.coli begins to form ℃ ℃

colonies, they were able to retain the LB AMPs adaptation and grow in ampicillin, an anti-

bacterial compound.

The second Step of the experiment was to do a PCR using Taq Polymerase to allow the 2

different Genes to anneal to each other under extreme temperatures. The intermediate steps

where to transfer 1.5 mL of the culture to a centrifuge test tube and put it into a 12000gs for 10

minutes. Remove the supernatant DNA pellet from the waste liquid. Resuspend the bacterial

pellet in 100 µL of cold GTE. Lysis the cells with 200 µL and store on ice for 5 minutes. Add

150 µL of cold K, AC solution, vortex the tube and disperse K, AC solution through viscous

bacterial lysate and store on ice for 5 minutes. Centrifuge at 12000gs for 5 minutes and then

transfer the supernatant to a fresh test tube. Add 1 mL of 95-100% cold ethanol. Mix it by

inverting the test tube and storing it on ice for five minutes. Centrifuge at 12000gs for 1 minute

in a microfuge, remove the supernatant pellet of DNA. Gently add .5 mL of 70% cold ethanol

and centrifuge at 12000g for one minute in a microfuge. Remove the supernatant pellet. Allow

the pellet to air dry until all liquid is gone from the tube. Resuspend the pellet in 50 µL of TE

until it has a pH of 8. Add a color coded dye to the mixture. So that it will be easier to load the

samples into the electrophoresis gel. To create the gel, mix 1 gram of agarose powder, 50 mL of

TAE, and microwave for 30 seconds. Then add 2 µL of ethmid bromide, put it in a minus 20 ℃

freezer and wait for 10 minutes. Lastly, load the gel into TE, load the DNA samples and run for

an hour. Once the hour is up, look at the samples under ultra violet light and look for solid bright

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orange bands. If the bands are solid then DNA was able to anneal, If the samples appears

striated, or empty then the primer tried to anneal and failed or the primer could not find anything

to bind to.

For the creation of the master mix with taq-PCR, water, 10x Buffer, 2.5 mM dNTP,

Primers 1 and 2, DNA from the E.coli pellet, and the taq Polymerase. First the master mix has to

be made in order to add the DNA samples. For water, 331.2 µL is added to a centrifuge test tube.

Next, 45 µL of the 10x Buffer is added to the test tube, and lastly, 36 µL of 2.5 mM dNTP are

added. For the creation of the primer, 1 µL of the 3 prime and 5 prime ends need to be put into a

centrifuge test tube. Next, 2 µL of DNA is mixed in. Lastly, 0.2 µL of the taq polymerase is put

in. Again invert the contents to assure proper mixture of the compounds. After which the master

mix and the taq mixture is to be combined.

The next step is to create the agarose gel to do an electrophoresis of the tac-PCR. One

gram of agarose powder needs to be weighed out. Then add the powder to 50 mL of xTAE

solution and microwave it for 30 seconds. Open the door periodically so that the bubbles do not

overflow. Next, add 2 µL of ethmid bromide to the solution and mix for 10 seconds. Lastly, put

the solution into a gel tray and have it solidify in a -20 freezer for 10 minutes. After the 10 ℃

minutes are up cut the gel out of the stencil, submerge the gel block into an electrophoresis unit

and set it for an hour. The following results below are from the E.coli culturing using the LB

AMP medium.

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Figure 1: Su(z)-12 (Not I) E.coli growth after one day incubation at 37℃

Only one colony formed which could mean that there was an error or contamination.

Figure 2: Jaz-f1 (Not I) E.coli growth after one day incubation at 37 ℃

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Remove the colonies before they destroy the AMP membrane so different E.coli strains do not

form.

Figure 3: First trial for the cancer fusion PCR

Legend: First port is the ladder, (3,4) and (6,7) hold the Su(z)-12 and Jaz-f1

The DNA did not anneal to create the fusion gene. This is believed to have happened due to a

pipette measuring error which made it so not enough DNA was present for the primers to find

and anneal to it. Either that or there was enough DNA but the temperature was not good enough

to allot for proper annealing to occur. To compensate for this, the next trial was done at multiple

annealing temperatures over the course of 5 hours to give the highest likelihood of DNA

annealing. The master mix was redone and the agarose gel was remade. There was an error in the

creation of the master mix. There was a mix in the numbers with the amount of water put into the

mix. In the second trial, only 313.2 µL instead of 331.2 µL of water was used. This error

however did not skew to the results in any way because it only increases the probability of

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detection of the DNA. Only one sample out of the 8 was put through the electrophoresis without

the master mix.

Figure 4: Second trial for the cancer fusion PCR

Legend: First port is the ladder, (2-5 is the Su(z)-12) and (7-10 is the jaz-f1)

After the second trial of experiment 1 there was a positive identification of 2 mutant DNA bands.

After this positive result, the gel was resubmerged into the TAE solution and put under

electrophoresis for another 30 minutes. After the 30 minutes was up, the gel was reexamined

under UV light and both bands had disappeared. This was not normal and rarely happens if it has

ever happened before. There are a few possible reasons that could explain this phenomenon.

First, when submerging the gel back into the TAE, the influx of solution could have pushed the

DNA out of the ports. The next hypothesis is that when it underwent the elextrophoresis the

change in voltage may have disrupted the DNA somehow. During the first electrophoresis, the

voltage was at 105 volts. During the second run the voltage was 56 volts. This might have

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disrupted the DNA. The last hypothesis is that there was never a DNA to begin with and that UV

light had given the illusion of a sample being show. Either way it is uncertain.

Conclusion: The experiment yielded no valuable results for the creation of the mutant

gene of Su(z)-12 and Jaz-f1. This can be attributed to human error due to pipetting measurements

being off. Laos the temperature may not have been high enough to allow for annealing. The last

step is to then cycle them through various temperatures to encourage annealing of the DNA. First

the temperature will be put at ninety-four degrees Celsius to weaken the hydrogen bonds of the

DNA to allot for a higher success of annealing by opening up its helix. After this the temperature

is cooled to the annealing temperatures of fifty degrees Celsius for five cycles, fifty-five degrees

Celsius for five cycles, and sixty degrees for another twenty-five cycles. This process took

upwards of five hours to complete.

The second trial of the experiment went exceedingly well due to getting two positive

results, but due to a unfortunate occurrence they had disappeared without any reason why. The

experiment was not a failure however, the second trial of the experiment re-proved that it was

possible to transform the plasmids and drag the gene along. Also, the fact that the DNA from the

second experiment went missing beckons many more scientific questions. This experiment

shows that even in a controlled environment the most peculiar of events still happen both good

and bad. A method that could be better than doing a PCR could be to forcibly splice the two

genes together that way it is not a matter of chance, but a matter of how many attempts it takes.

This can also be an avenue to test what the fusion gene can do if spliced in different genes. For

example if the fusion gene caused a benign tumor in the thorax of Drosophila melanogaster, it

would be reasonable to suggest that a fusion that has the power to kill, also has the ability to be

inert if ended up in that alternate gene. The results from the mouse experiment at consistent with

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the experiment done. Even though the mutant could not be created, it was still possible to

transfer the DNA plasmids and get a short term positive result on the creation of the mutant gene.

References

The JAZF1/SUZ12 fusion protein disrupts PRC2 complexes and impairs chromatin repression during human endometrial stromal tumorogenesis. (n.d.). Retrieved December 03, 2016, from

https://www.ncbi.nlm.nih.gov/pubmed/27845897