FREE ESSAY ON THE IDENTIFICATION OF AN UNKNOWN PLASMID USING RESTRICTION ENZYMES, GEL ELECTROPHORESIS, AND TRANSFORMATION.

THE IDENTIFICATION OF AN UNKNOWN PLASMID USING RESTRICTION ENZYMES, GEL ELECTROPHORESIS, AND TRANSFORMATION.

The Identification of an Unknown Plasmid using Restriction Enzymes, Gel Electrophoresis,
and Transformation.
Abstract
Using DNA technology, including restriction enzymes, gel electrophoresis, and
transformation we performed two experiments to identify an unknown plasmid. The first
experiment involved gel electrophoresis of plasmid DNA with and with out restriction
enzymes to determine migration and number of base pairs in specific fragments. Known DNA
fragments were used to determine the size of the unknown fragments. Plasmid maps of pKAN
and pAMP were used to compare the sizes to the unknown sizes, where by identification of
the unknown was pKAN. The second experiment used the same unknown plasmid in a
transformation to bacteria E.coli HD . The recombinant DNA of the bacteria was
analyzed by growing the bacteria in plates containing antibiotics. The growth of the
unknown sample was shown only in the pKAN/LB plate and the LB (standard medium) plate,
thus the bacteria contained DNA which held resistance to kanamycin, a characteristic of
the pKAN plasmid. Through the results of both experiments we therefore conclude that the
unknown plasmid was pKAN. 
Introduction
Recombinant DNA is the extracting DNA from one genome and incorporating it into another
genome. This genetic technology is used in a variety of ways and benefits a variety of
people and industries. The usage of recombinant DNA makes products like insulin for those
who are diabetic. Other health related uses include synthesis of vaccines and
antibiotics, and research is currently being conducted to further integrate recombinant
DNA into the world of medicine and health. Another important use of recombinant DNA is in
agriculture. Both animal and crop agriculture benefit from this technology, plants can be
created with greater pest resistance and higher quality, and animals can be injected with
hormones synthesized through recombinant DNA to help regulate and improve their growth
and reproduction. DNA technology has hundreds of applications, thus has its place firmly
established in the realm of science (1). 
Recombinant DNA allows scientists to isolate DNA, transport it to other organisms, and
study the effects in the DNA in a new organism. This is done using enzymes to cut the DNA
into fragments. The enzymes used are called restriction enzymes, which are those enzymes
that selectively recognize and degrade foreign DNA. Selected fragments of cut DNA are
then placed into a vector to carry foreign DNA sequences from one host cell to another.
The small, circular DNA molecules found in bacteria, called plasmids, are a common vector
for this technique.(3). Plasmids generally replicate independent of the chromosomal
replication and are very useful for replicating the desired DNA quickly. Once the DNA is
acquired in the plasmid, it is ready to be placed into an organism (2). 
The bacterial mechanisms of genetic recombination include three processes; conjugation,
transformation, and transduction. The most common of these mechanisms in laboratories is
transformation. Transformation is the alteration of a bacterial cell's genotype by the
uptake of naked, foreign DNA from the surrounding environment. Conjugation is the
transfer of genetic material between two bacterial cells that are temporarily joined by a
pilus. Transduction is the the transfer of genes from one bacterial cell to the other by
a bacterial virus called a bacteriophage. Once the genetic recombination takes place,
observation of the phenotypical changes produced by the recombinant DNA can commence
(4).
Once an enzyme has digested DNA, its changes can be viewed using gel electrophoresis. Gel
electrophoresis incorporates the fact that DNA is a negatively charged molecule. Samples
are placed into a agarose gel well where they are moved through the gel in an electric
field. During the electrophoresis, the DNA fragments are sorted by size. The smaller DNA
fragments migrate farther than the large fragments. When DNA is treated with a
restriction enzyme, it is possible to observe the effect of the enzyme using the gel
electrophoresis technique. Depending on the type of enzyme and the type of DNA, the
number and shape of DNA fragments can change. In a plasmid, change in shape is due to the
original supercoiled shape of the plasmid becoming transformed into linear pieces or a
circular shape. The supercoiled shape migrates the farthest, followed by the linear
shape, and then the circular shape. The change in the fragments can be detected using a
staining solution like EtBr on the gel and exposing the gel to an ultraviolet light (4).

The purpose of these experiments is to identify an unknown plasmid. Plasmids often carry
genes for antibiotic resistance, and our unknown plasmid could come from either of two
plasmids carrying antibiotic resistance. pKAN has a resistance gene for kanamycin and
pAMP has a resistance gene for ampicillin. We are given plasmid maps of each plasmid, and
these tell us the numbers of base pairs in each fragment when cut with specific enzymes.
We use gel electrophoresis to study the effects of the restriction enzymes on the plasmid
and use the data we gain to compare it to the plasmid map, therefore helping us to
identify the plasmid. We observed the DNA fragment of the uncut, singular, and double cut
unknown plasmid and also compared those to a standard DNA fragment cut in multiple
places. The second experiments purpose is to also identify the unknown plasma, but this
time we use recombinant DNA technology to conclude our identification of the unknown
plasmid. Given the unknown plasma, we use transformation to incorporate the genes into
bacterial cells. We can then specifically look at the effects of the foreign DNA on the
host organism. 
In Experiment 1 we observed the Standard DNA piece cut in several places with a
restriction enzyme lambda Hind III, and we hypothesize that we would obtain several
fragments upon Gel electrophoresis. The number of base pairs in each fragment was given
and by measuring how far the pieces migrated we hypothesized that we could determine
sizes of the unknown plasmid DNA pieces in relation to how far those pieces migrate. The
second sample was U (uncut sample) of our unknown plasmid. No enzyme cut this sample,
therefore we predicted there would be only one fragment shown in gel electrophoresis.
This sample was in the supercoiled form and we predicted that it would migrate farther
when compared to the S cut. The next sample was an S (single cut) sample of the same
unknown plasmid. This sample of DNA was cut once with a restriction enzyme Hind III and
therefore we predicted the shape would change to a linear fragment of DNA. The final
sample was a D (double digested) sample of our unknown plasmid. This sample was cut with
the restriction enzymes Band H1 and Hind III. Since the sample was cut twice, we
hypothesize that it will show two DNA fragments. We further hypothesize that we will be
able to determine the unknown plasmid based on the size of the fragments, as the enzymes
Hind III and Bam H1 cut pKAN and pAMP at different places producing different size
fragments. Using a plasmid map, we predict that we will identify the plasmid based on the
data obtained from the gel electrophoresis.
Determining unknown plasmids using gel electrophoresis and plasmid maps is very important
for genetic engineering. DNA technology makes it possible to clone genes for basic
research and also has many commercial applications. The toolkit for this technology
includes restriction enzymes, DNA vectors (like plasmids), and the gel electrophoresis
techniques to separate and identify the DNA fragments. 
In Experiment 2 we were given four microfuge tubes containing DNA and another tube of
cells E. coli DH5. Of the four tubes, one was the unknown plasmid DNA from
Experiment 1. The positive control was the DNA with known plasmid DNA in them. One
positive control contained the DNA of pAMP and the other contained the DNA of pKAN. The
negative control contained a buffer solution in the tube with no DNA present. After
transformation was complete in the E.coli DH5, the cells were then cultured onto
three different plates. One plate contained a standard growth medium (LB), the second
contained LB and the antibiotic ampicillin, the third contained LB and the antibiotic
kanamycin. We hypothesize that the bacteria which have the plasmids containing the gene
for antibiotic resistance will be able to grow in the plates containing that antibiotic.
We also predict that we can identify the unknown plasmid by which type of environment it
grows in. We expected that the negative control would only grow in the standard medium
since there was no DNA to transfer in that sample and no antibiotic resistance given.
In biotechnology, the technique of transformation is applied to introduce foreign genes
into bacteria. These genes code for proteins that can be very valuable. For example,
human insulin and growth hormone are synthesized using this technology.
Materials and Methods
In Experiment 1 we prepared our 8% Agarose Gel to be used in gel electrophoresis. We
placed the buffer tray, the dams, and the comb into the gel box and poured 30 mL of the
Agarose solution into the tray. The solution was left to harden for 30 minutes.
We were given four microfuge tubes containing our unknown plasma. Three of them were the
three different treatments of our unknown plasmid. Treatment U was uncut and in its
natural state (supercoiled). Treatment S was a single digest of our unknown plasmid, cut
by enzyme Hind III. Treatment D was a double digest of our unknown plasmid, cut by Hind
III and Bam H1. The final tube was of a standard DNA sample digested with lambda Hind
III. Given data about this standard DNA, we used it to compare the unknown fragments to
the known fragments. We added 2L of loading dye to each of the four tubes, using
a Wiretrol microcapillary tube. Next, we placed the tubes in the microfuge to force the
dye into the bottom of the tube, pulsing for 5 to 10 seconds. We then measured 150 mL of
Tris borate EDTA buffer and poured this into the trough of the electrophoresis box. Next,
we loaded the samples into the agar well in the electrophoresis box using the hand held
pipettor. We turned the power supply on and set the dial to generate 110 volts. We
stopped the electrophoresis process at 30-45 minutes, when the first blue dye streak
reached the last red band on the electrophoreses box. The gel was then stained for 10
minutes in ethidium bromide, then placed on the UV transilluminator and observed. A
photograph was taken of the gel.
For Experiment 2, we were given four microfuge tubes with DNA samples and a sample of the
competent cells. One of the positive controls, labeled PA, contained pAMP DNA, the DNA
that had the gene for resistance to ampicillin. The other positive control, labeled PK,
contained pKAN DNA and had the gene for resistance to kanamycin. The negative control,
labeled TE, contained only the buffer and no DNA. The last tube contained DNA from the
unknown plasmid of Experiment 2. We used a hand help pipettor to transfer 100L of
cells to each DNA microfuge tube. We capped and finger vortexed (mixed) the cells and
DNA. The tubes were then incubated on ice for thirty minutes. The tubes were then heat
shocked for 45 seconds in a 42 C water bath. The tubes were rapidly transferred to the
ice for another two minutes. Using a 1 mL pipette, 0.9 mL of LB broth was transferred to
each of the microfuge tubes. The samples incubated for an additional 60 minutes in a 37 C
water bath.
For each of the four tubes, 100L of sample was transferred to each of three
different agar plates. The plates were LB; a nutrient-rich standard medium containing no
antibiotic, LB/AMP; the standard medium containing the ampicillin antibiotic, and LB/KAN;
the standard medium containing the kanamycin antibiotic. The cell/DNA samples were
cultured on the plate using the confluency streaking method, and incubated at 37 C. 
Results
In Experiment 1, we had results that identified our plasmid as pKAN. Our electrophoresis
gel photograph showed that the standard DNA sample (digested by lambda Hind III)
contained many bands. This is what we expected as this standard DNA has many sites for
digestion of the enzyme, producing many fragments. We were given the size of the standard
fragments in numbers of base pairs, and we then measured the distance that each fragment
traveled. 23,130 base pairs (bp) traveled 13mm, 9,416 bp traveled 16mm; 6,557 bp traveled
20mm; 4,361 bp traveled 22mm; 2,322 bp traveled 25mm; 2,027 bp traveled 30mm. Using the
size of the fragments in the y-axis and the distance migrated in the x-axis, we
constructed a graph to compare unknown fragment migration to size of fragment in base
pairs (figure 1). This graph shows the proportional relationship of smaller size DNA
migrating farther down the gel.
The uncut sample (U) showed two fragments of DNA at 23mm and one at 27mm. This is not
what we expect as the sample is supercoiled and has no cut fragments. The single cut
sample (S) showed one fragment at 24mm. This is because the single cut produced a linear
DNA fragment which is what we expected. The double cut sample (D) showed two fragments,
and they measured 30mm and 32mm. This is what we expect as the fragment has been cut into
two pieces. The attached photograph of the gel electrophoresis shows the U sample being
slightly further ahead than the S sample, and this is expected as the U sample is
supercoiled.
Using the graph obtained from the standard DNA sample, we found that migration at 21mm
and 33mm corresponded to approximately 2,100 bp and 1,500 bp. Using the plasmid map we
found that the sizes of fragments of our unknown were close in size to the fragments of
pKAN, thus we identified our unknown as the plasmid pKAN.
For experiment 2 we found that the bacteria cells did grow from the PA sample cultured in
the LB/A plate and not the LB/K plate, this is predicted as our positive control. Growth
was shown from the PK sample, only on the LB/K plate and not on the LB/A plate. This is
also another positive control. Both the PA sample and the PK sample showed growth on the
LB (pure standard medium) plate. The negative control, TE with no DNA, did not show any
growth on the plates LB/A or LB/K. This is what we expect of our negative control because
the TE sample had no DNA transformed into the bacterial cell to give it antibiotic
resistance. The TE sample did have growth on the LB plate, this is expected because the
bacteria were not in an environment of antibiotics. The unknown sample showed growth on
the LB/K plate and no growth on the LB/A plate, and there was growth on the LB plate.
Because the bacteria grew in an environment with kanamycin, it is likely that the pKAN
plasmid was the unknown DNA plasmid. We conclude again, that our unknown plasmid was
pKAN. 
Discussion
In experiment 1 we used restriction enzymes, gel electrophoresis, and plasmid maps to
identify the unknown plasmid. This was done by observing standard DNA digested by lambda
Hind III, and plotting its known size of fragments with the migration distance shown
through gel electrophoresis. It is known that DNA can be cut by enzymes and the fragments
of the DNA will migrate according to size in the gel electrophoresis. By creating a graph
(figure 1) we were able to compare the migration of the unknown plasmid fragments and
determine a size for the fragments. It is also known that through the restriction
enzymes, the DNA shape can be altered and thus the speed at which the fragment travels
can be altered. We hypothesized that the uncut DNA being supercoiled would migrate faster
than the single cut DNA. This was observed, as the uncut DNA did move slightly farther
than the single cut DNA which was in linear form. Although both fragments have the same
number of base pairs, the supercoiled form allows for greater speed in migration. The
linear form does not travel as fast as the supercoiled form. The double cut fragments
moved farther than the uncut or single cut because the D fragments were smaller, and
smaller fragments move faster and thus father during electrophoresis. 
It was also hypothesized that the double cut DNA would give us two fragments in linear
form. These fragments size were based on where the enzyme cut the DNA, and would be
different for the different plasmids pKAN and pAMP. We hypothesized that we would be able
to determine the unknown plasmid based on the size of the fragments using the plasmid
map. It was observed that the double cut DNA provided the fragments to compare with the
graph and plasmid map, and ultimately identify the unknown plasmid. The double cut
fragments migrated 21mm and 33mm. When analyzing this data, we used the graph to
determine the approximate sizes of the fragments from the D sample. We found that 21mm
corresponds to approximately 2,100 base pairs (bp) and 33mm corresponds to approximately
1,500 bp. Then we used the plasmid map to determine which plasmid DNA we had in our
sample. The plasma map for pAMP showed that the enzymes used to cut the DNA (Hind III and
Bam H1) cut at 1120 bp and 1904 bp, this would leave DNA fragments at 784 bp (1904-1120)
and 3755 bp (4539 (total bp) - 784 bp). Our data does not correspond to this plasmid. We
used the same technique to analyze the plasmid map of pKAN, its fragment sizes were 1875
bp and 2332 bp when cut with the enzymes Hind III and Bam H1. These DNA fragments are
close in size to our unknown plasmid DNA fragments, thus we concluded that our plasmid
DNA is pKAN. 
In Experiment 2 we identified the unknown plasmid using recombinant DNA through
transformation of plasmid DNA to E.coli HD. We used the same unknown plasmid as
in Experiment 1. It was known that bacteria take up foreign plasmid DNA into their cells
through transformation and exhibit characteristics of the foreign DNA. In the positive
control PA, the results were that bacterial growth was observed on the LB and the LB/A
plate. There was no growth on the LB/K plate. The LB plate had no antibiotic in its
environment, so we predicted that all the bacteria samples would grow in this medium, and
all did. The LB/A plate contained ampicillin in its environment, and we hypothesized that
the PA (bacteria with plasmid pAMP) group would be resistant to this antibiotic and grow
normally. This is was what observed. We also hypothesized that the PK group would grow in
the LB and LB/K plates, as the PK group contained bacteria cells that have the gene
responsible for resistance to kanamycin. This was observed as the PK group grew in the
environment containing kanamycin (LB/K), and did not grow in the environment containing
ampicillin (LB/A). Thus, all our positive controls did work. 
The negative control TE worked as well. Not having any DNA in the solution during
transformation, we predicted that no bacterial growth would be observed in the LB/A and
LB/K plates. This is because the genes for resistance are not present. The TE group did
show growth on the LB plate, signifying that the bacteria were present and proliferating
in this group. Growth was possible in this medium because no antibiotic was present. 
We hypothesized that we could identify the unknown plasmid by observing the growth of the
bacteria on antibiotic plates. If there is growth on a plate containing an antibiotic we
assume that the bacteria has taken up, through transformation, the plasmid containing the
gene for resistance for that antibiotic. This is what was shown as the unknown sample did
show growth in the LB and the LB/K plates. The results show that the unknown plasmid was
pKAM because bacterial growth was possible on the LB/K plates where kanamycin was
present. There was no growth on the LB/A plate from this group. 
From the two experiments, one studying the genotype of the plasmid and the other
observing the phenotype, it is conclusive that the unknown plasmid in these experiments
was pKAM.
Both experiments went relatively well, as we had little problems when performing these
experiments. In experiment 1, better technique in loading the samples so as not to break
the gel or miss the well would have been helpful, but this is minor, as practicing would
contribute to effective technique. 
Bibliography
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2.) Campbell, Neil. 1996. Biology, 4th edition. p.338-339, 369 Benjamin/Cummings
Publishing Co., Inc. New York.
3.) Johnson, Shea. Recombinant DNA. Student Reports of BI 220 University of Oregon.
Infoseek. http://ponderosa-pine.uoregon.edu/Bi220/Johnson/menu.html
4.) Lawrence, S.M., M.K. Heidemann, and D.O. Straney. 2000. Biological Sciences 111L
Laboratory Manual, 4th edition. p.141-148