Check out the video I made for my biology class regarding DNA extraction through ethanol precipitation!
http://www.youtube.com/watch?v=UQQOwRPYvzg
pGLO Bacterial Transformation
Monday, 30 April 2012
Friday, 9 March 2012
Beta Vulgaris Root Tonoplast and Cell Membrane Permeability Increase Through Immersion in Solutions of; Low pH, Extreme Temperature, and of High Solvent Concentration.
Beta Vulgaris Root Tonoplast and Cell Membrane Permeability Increase Through Immersion in Solutions of; Low pH, Extreme Temperature, and of High Solvent Concentration
Eric Horton
March 5th
2012
SBI 4U1 – 70
Prepared for
D. Melegos
Abstract
This
manuscript explores the affects of various stresses on the permeability of cell
membranes and tonoplasts. Some background information will be provided covering
various topics including the fluid mosaic model, free radicals, antioxidants,
betanin, spectrophotometry and Beer’s law. The experimental approach for the
lab will be highlighted along with the objectives and anticipated results. A
clear outline of the methodology followed will be covered followed by the
results obtained through the lab. The results will be explained along with
sources of error present during the lab and future lab prospects.
In order to understand the logic behind
the methodology of this lab, and understand the results, it is important to
have a firm grasp of a few preliminary concepts. For example, the fluid mosaic
model, seen as figure 1 in the
appendix section, is the most widely accepted model used to describe the
composition of biological membranes. The term mosaic implies the nature of the membrane, showing how it is
composed of many different structures that work side by side without creating
an intermediate substance [1]. In general, the structure of
biological membranes consists of a lipid bilayer, with impeded proteins [1].
The term fluid mosaic implies the presence of lateral motion within the membrane.
The proteins float in the lipid bilayer, free to move along the plane of the
membrane [1]. Other components of the membrane include
oligosaccharide chains, membrane proteins and cholesterol [1]. Understanding
the composition of the cell membrane is key to understanding what stresses will
be the most successful in altering the permeability of the Beta vulgaris’ cell
membranes.
Free radicals are very unstable molecules
that possess an odd unpaired electron. This is a result of a bond splitting,
leaving the odd unpaired electron as depicted in figure 2. As a result of their unstable nature, free radicals react
quickly with other compounds attempting to capture the needed electron,
regaining stability. This means that free radicals tend to attack the nearest
stable molecule in order to “steal” an electron, leaving that molecule as a
free radical. This creates a chain reaction that eventually can disrupt the
living cell. Free radicals can be created by your body naturally during
metabolism. Sometimes the body’s immune systems cells purposely create free
radicals in order to neutralize viruses and bacteria present in the body. On
the other hand, environmental factors the body is exposed to such as pollution,
radiation, cigarette smoke and herbicides seen in figure 3 can cause free radical formation. Interestingly enough,
Free radical damage tends to accumulate with age. [2]
Antioxidants
are substances such as vitamin C or E that remove potentially damaging
oxidizing agents from a living organism. Vitamins C and E are thought to
protect the body against the damaging effects of free radicals. These
antioxidants neutralize free radicals by donating one of their own electrons.
This puts an end to the “electron stealing” chain reaction created by free
radicals. Antioxidant nutrients don’t become free radicals themselves after
donating an electron because they are stable in both forms. Their unique
properties help prevent cell and tissue damage that could lead to disease.
Vitamin E tends to protect against cardiovascular disease by defending against
LDL oxidation and artery-clogging plaque formation. Many studies have linked
high vitamin C intakes with lower rates of cancer, particularly cancers of the
mouth, larynx and esophogus. [2]
Betanin is a food additive used to give
a red colour to many food products including meat, ice cream and various
beverages. Betanin is found in red beats or Beta
vulgaris and is a type of antioxidant. Being an antioxidant, the
consumption of betanin provides many benefits against oxidative and stress
related disorders. Betanin is also known to increase the red blood cell count
in the human body and maintain a proper metabolism. The flow chart labelled
figure 5 in the appendix section shows the complex series of steps followed
during the biosynthesis of betanin. Betanin has a structure as seen in figure 4, which consists of a great deal of nitrate, which can also be
hazardous to human health in high doses. [3]
Spectrophotometry is the measurement of
a solutions colour by determining the amount of light absorbed in the
ultraviolet, infared, or invisible spectrum, used to calculate the
concentration of a substance in solution [4]. When a beam of
incident light passes through a solution, part of the light is reflected, part
is absorbed and the rest is transmitted. The relationship that exists between
the concentration of a compound and the extent of absorption of light is based
on Lambert’s Law and Beer’s Law [5].
According to Lambert’s law, the light absorbed by a solution is directly
proportional to the length of the light path through the solution [5].
According to Beer’s law, the amount of light absorbed is directly proportional
to the concentration of absorbing solute in the solution [5]. During
this lab, spectrophotometry will be a key tool used to determine the relative
effectiveness of each stress on the Beta vulgaris’ cellular membrane.
The objective of this lab was to
investigate the extracellular concentrations of betanin, and correlate it with
the cell membrane/tonoplast permeability. Factors used to test the viability of
these cell parts will include the determination of the optimal pH, temperature,
and solvent. These results will be quantified using a spectrophotometer.
The approach taken with this experiment
involves various controls coupled with isolation of the various factors. In
order to test for the optimal pH value for cell membrane permeability, five
beet root disks were immersed for 10 minutes in a test tube containing one of
the six solutions, with pH’s of 2,4,6,7,10 and 12. Within this portion of the
experiment, water (pH of 7) acts as a control of sorts. This “neutral” pH
should have little to no effect on the membrane.
For the second portion of the
experiment, temperature was isolated as a factor affecting the membrane
permeability. Five different tests were run, one with five disks inserted into
a test tube with water at a temperature of -180 C, the second set in
a test tube with water at 40 C, the third set was placed in water at
210 C, the fourth set was put in water at 400 C and the
final five disks were put in water at 800 C. All of these solutions
were left to incubate for 10 minutes. The room temperature water in this case
(210 C) acted as a control.
For the final variable, five beet root
disks were put into each of the six test tubes containing various solvents of
varying concentrations for 10 minutes. The solutions used included; 1% acetone,
25% acetone, 50% acetone, 1% methanol, 25% methanol, 50% methanol.
After the incubation time for each of
the samples was complete, the extracellular fluid from each test tube was
poured into a small cuvette and then inserted into the spectrophotometer. The
resulting absorbance values were examined and a conclusion was drawn.
It is anticipated that the greatest
absorbance will occur when the cells have been submitted to the harsher
stresses. Various high and very low temperatures should return a high
absorbance value, along with the higher concentrations of solvents. High and
low pH’s should also cause greater cell membrane and tonoplast permeability,
resulting
in higher absorbance values. The more moderate conditions aren’t as likely to
increase the cells permeability and in turn should have low absorbance values.
Methodology
The methodology for this lab was broken
down into several steps. The first step involved the preparation of the Beta
vulgaris root for testing. The beet was prepared by slicing the top and bottom
off of a fresh beet. Using a cork borer, a core sample of the root was
extracted. After driving the borer into the beet, the tweezers were used to remove
the core sample. The cylindrical root section was then sliced into small 3mm
thick disks using a scalpel. For our section of the experiment, fifteen disks
were prepared. Once all the cutting was complete, the disks were put into a
beaker and then immersed in tap water. The beaker was then agitated, swirled
and then emptied. This process was repeated three times in order to adequately
rinse the contents of the burst cells from the samples.
In order to test the permeability of the
cells under various temperature conditions, five pre cut and rinsed disks were
placed in the freezer at a temperature of eighteen degrees below zero Celsius.
Five disks were also placed in the fridge at a temperature of four degrees
Celsius. Five disks from the freezer were then put into a large test tube, five
disks from the fridge were placed in another large test tube, and then the five
of freshly cut disks were placed into each of the three remaining test tubes. Using
a graduated cylinder, 10ml of tap water was measured out and added to each of
the five test tubes. Each of the samples was incubated for 10 minutes. The
samples containing the disks from the fridge and freezer were incubated at room
temperature along with the 210 C sample. The 400C and 800C
samples were incubated in water baths at their assigned temperatures.
In order to investigate the relationship
between membrane and tonoplast permeability and varying pH, a series of steps
were followed. As for the previous test, 25 3mm beet root disks were prepared
using the cork borer and scalpel. 5 disks were placed into each of the six
clean test tubes prepared. Each test tube was labelled using masking tape and a
permanent marker with the pH value of either 2,4,6,7,10 or 12. Then 10mL of
each solution of a varying pH was measured using a graduated cylinder, and then
added to the corresponding test tube.
As for the test involving pH, a key
sequence of steps was followed in order to test the effect of various solvents
of numerous concentrations on membrane and tonoplast permeability. 25 3mm beat
root disks were prepared using a cork borer and a scalpel. 5 disks were placed
in each of the 6 test tubes. Each test tube was labelled with one of the
following solvents and concentration; 1% acetone, 25% acetone, 50% acetone 1%
methanol, 25% methanol, 50% methanol. Then 10ml of each solvent was measured
using a graduated cylinder and added to the corresponding test tube.
All of the samples were then allowed to
incubate for 10 minutes, some at specific temperatures, and the rest at room
temperature. After the 10 minute incubation period, the samples were gently
agitated and then the liquid portion was poured into small test tubes. The
spectrophotometer was then set to 560nm and calibrated using a test tube
containing tap water. Once calibrated, each of the five small test tubes were
placed in the spectrophotometer and their absorbance readings were recorded in table 1 in the appendix section.
Results
After
conducting the above experiment, a clear and relatively definitive set or
results were obtained. As seen in table
1, it was clearly observed that as the different stresses were applied,
varying absorbance values resulted. With regards to pH, the lower acidic pH
values returned higher absorbance values than the moderate to basic pH
solutions. At a pH of 2, the highest absorbance value of 0.52 was observed
compared to the 0.02 value at the pH of water and 0.05 at a pH of 12. This
clearly indicates that the cell membranes and tonoplasts are significantly more
permeable in acidic solutions. As indicated by chart 1 found in the appendix section, a clear logarithmic trend
associating increasing pH with reduced absorbance values can be observed.
The temperature related stress
results were rather interesting as well. Table
1 indicates that the cell membrane and tonoplast are the most permeable at
extreme temperatures. At -180 C an absorbance value of 1.61 is
observed and at 800 C an absorbance value of 1.05 results. These
values contrast the absorbencies indicated at a more moderate temperature of 210
C where absorbance values of 0.04 were observed. Through plotting this
data on chart 2, a parabolic trend
is observed, this indicates that extreme temperatures provide the most stress
to a cells membrane and tonoplasts.
In the final test, it was noted that
with increasing the concentration of solvent used in the test, greater membrane
and tonoplast permeability was achieved. The absorbencies for this test ranged
from 0.02 for the 1% acetone solution, to 0.46 for the 50% acetone solution. For
the methanol, 0.02 absorbance values was observed at a 1% concentration, and at
a 50% concentration a 0.32 absorbance value was found to exist. Through the
exponential trends illustrated on chart
3 it became clear that not only as the concentration increased the
absorbency increased, but the acetone was more effective than methanol at
creating permeability.
Discussion
The
results obtained through the series of test conducted can be justified using
some basic knowledge of cell membranes. The results of the temperature portion
of the lab can be explained using the properties of cell membranes. When the
cell is submitted to high temperatures, the phospholipids become increasingly
fluid, increasing the permeability of the membrane. This allows materials
previously unable to travel in and out of the cell to do so. The extremely high
temperatures can also break the hydrogen bonds found in the protein structure
which could cause protein de-naturation. When the cell is frozen, the membrane
bursts because of the ice crystals and extreme temperatures make the cell
brittle, causing it to fracture. This allows an increase in permeability as
well.
When subjected to a solution consisting
of a solvent the membrane also becomes more permeable. Acetone and methanol are
both polar solvents. The phospholipids bilayer is also polar, meaning they will
tend to combine easily. Solvents denature the proteins found in the
phospholipids bilayer, meaning the sulphide bridges between cysteine amino
acids found in the quaternary and tertiary may be disrupted. The alteration to
the proteins structure stops them from being able to function correctly. This
protein alteration creates gaps in the cell membrane that allow for increased
permeability. As the concentration of the solvents increased, this effect is
further magnified. Acetone has a greater effect on the cell membrane because
acetone has a greater molecular dipole (2.91) than that of methanol (1.69). The
difference in polarity makes acetone a stronger solvent, resulting in a more
significant effect on the cell membrane.
pH also has a similar affect on the cell
membrane. With the increase in acidity during the pH stress test, protein
denaturation occurs as well. The change in pH tends to disrupt the ionic bonds
found in the proteins tertiary and quaternary structures. These disruption
cause changes to the shape of the protein and as a result of this the protein
is denatured. Changes to the proteins composition can cause an increase in the
membrane permeability.
Along
with the increase in permeability through each of the stresses, comes an
increase in the absorbency. As the cell membrane becomes increasingly
permeable, the more betanin is released into the extracellular fluid. With the
increase in betanin release, there is an increase in the absorbency measured
using the spectrophotometer.
Through conducting this lab several
sources of error became evident. During the temperature stress test, the
incubation time was supposed to be 10 minutes. However, by the time the water
was added to each of the test tubes, and each test tube made it to its
designated temperature environment, the times were skewed. With the present lab
set up it was impossible to incubate each of the samples for the same period of
time. The difference in incubation time could alter the absorbency values
significantly. This could be remedied by setting separate timers for each of
the test tubes incubation. Another source of error that became evident was the
insertion of the frozen or chilled beet disks into room temperature water. This
would moderate the temperature during incubation, rather than having a
consistent temperature. This problem however is somewhat unavoidable, unless
that water was made the same temp as the disk. This would pose a problem for
the frozen disk test however. The final source of error that was evident during
the lab was the consistency in the size of beet disks used. There was no step
taken to ensure absolute accuracy with respect to disk size. Different disk
sizes could contribute to large or fewer available cells and therefore lesser
or greater absorbency values. This problem could be fixed using a tool that
cuts consistent disk sizes. An example of this may include a cheese slicer. A
consistent disk size would make for far more accurate results.
A potential lab proposal may be one that
incorporates a comparison between polar and non polar solvents. Despite its
carcinogenic nature, benzene would be a good non polar solvent that could be
compared to acetone and methanol. 3 test tubes, one with 1% concentration the
others 25% and 50% respectively. The results of this test could be graphed with
the results of the polar solvent test. This would give insight into the effects
of the disruption of the London forces between non polar amino acids. Another
potential lab proposal might involve the isolation of betanin in order to
explore its abilities as an antioxidant. One might consider introducing free
radicals into a group of plant or animals and observing the effects. Then a
comparison might be made with the results after antioxidants are added to the
samples. This would allow us to verify and explore the importance of
antioxidants in our diet.
References
[1] Campbell, M. K., & Farrell, S. O. (2011).
Lipids and Proteins are Associated in Biological Membranes. Biochemistry
(7th ed., pp. 207,208). Belmont: Brooks/Cole.
[5] Chauhan, B. (2008). Principles of Biochemistry
and Biophysics. Bangalore, New Delhi: University Science Press.
[2] Understanding Free Radicals and Antioxidants.
(n.d.). HealthCheck Systems. Retrieved March 3, 2012, from
http://www.healthchecksystems.com/antioxid.htm
[3] Walker, M. (2011, February 25). What is Betanin
Used For? Health Benefits and Side Effects of Betanin. Kay Circle | Everyday
Reference. Retrieved March 3, 2012, from http://www.kaycircle.com/What-is-Betanin-Used-For-Health-Benefits-and-Side-Effects-of-Betanin
[4] spectrophotometry - definition of
spectrophotometry in the Medical dictionary - by the Free Online Medical
Dictionary, Thesaurus and Encyclopedia.. (n.d.). Medical Dictionary.
Retrieved March 3, 2012, from
http://medical-dictionary.thefreedictionary.com/spectrophotometry
Sunday, 15 January 2012
pGLO Bacterial Transformation Using Calcium Chloride Transformation Solution and Heat Shock
IDC-4U5
co-authors : Katelyn Dixon
Eric Horton
Sharmarke Mohamed
Jordan Khan
Abstract
Genetic transformation occurs when
an organism is modified by the introduction of new genetic information which is
incorporated into the organism’s genome. Bacterial transformation is the
easiest type of genetic transformation to create in a lab due to the single
celled nature of bacteria. In this lab the engineered pGLO plasmid is
incorporated into E. Coli bacteria, and adds the genes which code for the
proteins GFP and beta lactamase to the modified bacteria’s genome. To see the
effects of this plasmid on the cells, bacteria treated with the plasmid were
grown on two separate agarose plates containing LB nutrient broth and
ampicillin, and another containing LB nutrient broth, ampicillin and arabinose.
To contrast these plates two more plates were grown, one with LB nutrient broth
and ampicillin and the other with only the LB broth, using cells that didn’t
contain the plasmid. To see the effects of the calcium chloride solution used
in the procedure another experiment was run under the same parameters, except
the calcium chloride solution was replaced with distilled water. The results
showed that when calcium chloride was used the plasmid was successfully
incorporated into the E. Coli’s genome, since the cells treated with the
plasmid fluoresced green under ultraviolet light and were resistant to
ampicillin, an affect of beta lactamase. The results in the second test showed
that the calcium chloride solution is essential to efficient plasmid uptake,
but to what degree was not determined. Transformation efficiency was used in
this lab to quantify the uptake of the plasmid DNA. In the tests where calcium
chloride was used the transformation efficiency was much higher than those
where distilled water was used. In a further analysis of the experiment sources
of error, future prospects, and the ethical implications of bacterial
transformation are discussed.
Introduction
Bacterial
transformation occurs if a bacterium uptakes a piece of DNA and integrates it
into its genome. Bacterial transformation is done in two different ways in the
lab: electroporation and through calcium chloride/heat-shock. Electroporation is
when electrical shock is used to make the cell membrane permeable to DNA.
Calcium chloride/heat-shock is when the plasmids are incorporated into the
chemically-competent cells made permeable by the heat shock and calcium
chloride solution. In 1928, Frederick Griffith, a physician from London, was the
first person to experiment with bacterial transformation. He permanently
transformed a safe, nonpathogenic bacterial strain of pneumococcus into
a deadly pathogenic strain.[6]
Escherichia coli or E.
coli is a gram negative, bacillus, bacteria. E. coli makes a great bacterial
candidate for the bacterial transformation because it is made up of one cell,
it reproduces every twenty minutes, it does not harm people, and it cannot
survive outside of the lab. Ampicillin is an antibiotic that has a beta-lactam structure. It has the ability to kill E. coli cells. [7]
Plasmids are small
circular autonomously replicating pieces of DNA, found inside prokaryotic
bacterial cells. They replicate their own DNA by borrowing the cells’
polymerase. Due to their size, plasmid DNA is easy to extract and purify from
bacterial cells. Plasmids may express antibiotic resistant genes or be modified
to express proteins of interest, and are useful for cloning foreign genes. [8]
The pGLO plasmid
contains the gene for green florescent protein (GFP) and a gene for ampicillin resistances
called beta-lactamase . The pGLO plasmid also contains a special gene regulation system, or operon, that can be used to control expression of the florescent
protein in the transformed cells called araC regulator
protein. [6][7]
Green fluorescent
protein, or GFP, is found naturally in the luminescent jellyfish Aequorea Victoria.
GFP glows bright green when exposed to ultraviolet light due to resonation
which the light causes. GFP is mainly used in biotechnology as a biological
marker or indicator. It has also been used to produce luminescent plants and
animals.[4] GFP production is
monitored by a modified arabinose operon located
on the pGLO plasmid.
In the operon, the protein araC blocks
RNA polymerase from binding to the Pbad promoter. When arabinose is
present it changes the shape of the araC protein
causing it to promote, instead of prevent, RNA polymerase binding. Once RNA
polymerase has attached to the promoter, transcription of the GFP gene begins
and continues until arabinose runs out.[9][3]
Ampicillin is an antibiotic that falls into the penicillin group of drugs. Ampicillin is
often used to fight bacteria in the human body, specifically various types of
infections. Such infections may include those caused
by bacteria, like ear infections, bladder infections, pneumonia, gonorrhea, and E. coli
or salmonella infection. Ampicillin’s ability to exterminate bacteria makes it ideal for this lab [10].
During the course of
this experiment, our objective was to manipulate a variable present within the
lab and determine what affect this change would have on the results of the lab.
Our particular objective was to determine whether or not the calcium chloride
transformation solution was a critical ingredient in influencing the uptake and
expression of the pGLO gene. In order to accurately test this variable, we performed the
complete pGLO lab
as instructed in the pGLO manual as a control, and then proceeded to manipulate the lab
through duplicating the lab setup, however using an altered transformation
solution. The calcium chloride was replaced with distilled water for the second
set of plates, in order to compare the expression of the pGLO gene
in each case. In the end there was a total of 8 petri dishes. The first four were prepared ahead of time, and filled
with a solution containing nutrient agar solution, as well as a combination of
various solutions. These solutions included LB broth, ampicillin and arabinose. Two of the plates, (LB/amp) and (LB/amp/ara) we covered
in a solution consisting of transformation solution, LB broth and pGLO plasmid
DNA. The other two, (LB/amp) and (LB) were covered in a solution consisting of
only the transformation solution and LB broth. These four plates acted as our
control. We anticipated that the LB/amp/ara plate containing the pGLO plasmid DNA would become fluorescent. The other four plates were
the exact same, except none of them were covered with a solution that contained
the calcium chloride; instead distilled water was combined with the LB broth
and pGLO plasmid
DNA. It was anticipated that in the plates not containing the transformation
solution, there would be no expression of the pGLO gene. This result is expected because in theory, the transformation solution
is an essential step for giving the bacteria the ability to take up the pGlo plasmid, since the solution makes the bacterial cell walls more
permeable.
Results
The lab results can be seen in the appendix section.
As seen in figure 1, the control test, the LB plate containing
the -pGLO sample,
bacteria grew evenly on the plate in the areas where it was spread with the
transfer loop. In the LB/amp plate treated with the -pGLO sample absolutely no bacterial growth was observed. In the LB/amp
plate containing the +pGLO sample
several large, yellow colonies appeared, some grouped in clusters and others
spread over the plate. In the LB/amp/ara plate with the +pGLO sample many yellow, differently sized colonies grew, which
fluoresced green under ultraviolet light.
Figure 2 contains the results for the variable test, where the transformation
solution was replaced with distilled water. In the LB plate containing the -pGLO sample, bacteria grew evenly on the plate in the areas where it
was spread with the transfer loop. In the LB/amp plate treated with the -pGLO sample absolutely no bacterial growth was observed. In the LB/amp
plate containing the +pGLO sample
a few small yellow colonies appeared, spread randomly across the plate. In the
LB/amp/ara plate
containing the +pGLO sample
no bacterial growth was observed.
When the
transformation solution was used, there were 492 transformants per micro gram, as seen in figure 2 of the appendix section. However, when the transformation solution was replaced with distilled water, there were 0 transformants per
micro gram, as seen in figure 1 of the appendix section.
Figure 3 of the appendix section shows the calculations for the transformation efficiency on the LB/amp plate where no transformation solution was used. There were 67 transformants per micro gram on the LB/amp plate.
Figure 3 of the appendix section shows the calculations for the transformation efficiency on the LB/amp plate where no transformation solution was used. There were 67 transformants per micro gram on the LB/amp plate.
Discussion
In the control lab a different
outcomes was observed in each of the four plates. In the LB/amp/arabinose agarose plate
containing the +pGLO sample,
fluorescent green colonies developed. This is because the gene which codes for
the fluorescent protein, GFP, is located near the beta lactamase gene
on the pGLO plasmid,
which protects bacteria from the antibiotic ampicillin. When the cell produced beta lactamase to
deactivate ampicillin,
the GFP gene was also transcribed, producing the fluorescent protein observed.
In the LB/amp plate containing the +pGLO sample white, non florescent cells were observed. While these
genes contained the pGLO plasmid and the GFP gene they could not express the GFP gene
because they were not grown in the presence of arabinose. While the presence of ampicillin causes
the cell to transcribe the beta lactamase and GFP genes, arabinose is needed to activate the GFP operon. Therefore without arabinose in
the agarose gel, GFP cannot be transcribed and the cells will not fluoresce.
In the LB/amp agarose plate treated with the -pGLO sample, no cells grew. This is because without the pGLO plasmid
and the beta lactamase gene the cells cannot deactivate the ampicillin in
the gel. Therefore all the cells were wiped out. In the LB plate containing the
-pGLO sample small colonies were seen spread over the entire
plate. Because there was no ampicillin to kill the cells, all the cells survived and the entire plate was
covered, in contrast to the other plates where
individual colonies represented the cells which had taken up the plasmid.
In the variable lab,
where the calcium chloride solution was replaced with distilled water, results
for the -pGLO samples
were the same. This is because the calcium chloride solution affects plasmid
uptake and no plasmid was introduced to these samples. On the LB/amp/arabinose plate containing the +pGLO sample no bacterial growth was seen. This is because without the
calcium chloride solution to make the cell walls of the bacteria more permeable
the plasmid was not taken up by the cells. Without the beta lactamase gene
to deactivate the ampicillin all the cells were killed and no colonies developed. In the LB/amp plate containing the +pGLO sample a small amount bacterial growth was seen. This means that
some of the bacteria was taken up by the cell, since the beta lactamase gene
prevented the ampicillin in the plate from killing all the bacteria cells. It is unknown
why there was no plasmid uptake in the LB/amp/ara plate but some in the LB/amp plate. It may be due to an inconsistency
in the procedure, or a random occurrence. Due to the varying results in this
test, the affect of replacing the calcium chloride solution with water cannot
be definitively stated, but it is known that it reduces plasmid uptake.
The transformation
efficiency of our control test was 492 transformants per
microgram. This is much lower than the average of between 800 and 700 transformants per
microgram. This lower number could be a result of sources of error that may be
present within the methodology of this lab or potential human error. Our
variable test had an even lower transformation efficiency of 0 transformants per
microgram. This could be attributed to the lack of transformation solution, but
the tests should be repeated to reach a definitive result.
Due to the multiple solutions and
bacterial plates used in this lab there it is likely that some cross
contamination occurred. Though many precautions were taken, such as using
disposable pipettes and sterile loops, there is always a chance that these
tools could be contaminated before use, or that a new substance, such as
bacteria, was introduced from the environment. While this could be improved by
using a culture hood or wearing gloves, cross contamination, especially from
the environment, can never fully be prevented.
In this lab transformation efficiency
was used to measure how successfully the plasmid was incorporated into the
bacterial cells. While calculating transformation efficiency it was found that
it depended highly on the amount of bacteria taken from the starter colony.
Because of this inaccuracy the success of the experiment cannot be accurately
quantitatively described. Even if only one colony is taken, it is unlikely that
any two colonies are exactly the same size.
The transfer pipettes used during much
of the procedure weren’t very accurate, since any variation of the pressure
applied to the pipette would change the volume. This inaccuracy could be
eliminated by using micropipettes, which are accurate to the microliter.
In the variable lab, the lack of
calcium chloride solution had different effects on both +pGLO plates. On the LB/amp/ara plate no plasmid was incorporated into the cells, but on the
LB/amp plate the plasmid was at least partially taken up by the cells. The
reason for this inconsistency is unknown but is probably due to some error made
in the methodology. This might have been because of cross contamination or
incorrectly timing the temperature shocks used. To determine the cause of this
anomaly the variable lab could be repeated to see if the results were similar.
This lab has many future prospects.
Genetic modification does not have to be limited to florescence. Organisms can
be modified to have all sorts of interesting and unique traits. For example,
plants can be given plasmids so that they gain certain traits, such as
resistance to disease or extreme weather. This can lead to better crop yield
and shelf life. A lab extension of the pGLO lab
may involve modifying more complex organisms than bacteria. For example, one
could attempt to influence the expression of the GFP gene in fish, or another
multi cellular organism. Another potential lab
extension might include the isolation and purification of GFP. This could
be done using column chromatography. It may also be worthwhile to repeat the
experiment without the transformation solution to ensure the results are the
same. This would further explain the effect of the calcium chloride solution.
There are many ethical
dilemmas associated with this lab because of the nature of this experiment.
During this experiment, living organisms are being altered. Many would argue
that in doing so the experimenters are “playing god”. This may seem like an
absurd thing to question, but this is a living creature that simply just has no
way of expressing itself to us. Do single celled organisms have fewer rights
than us? Why should we be able to grow and kill these organisms at our own
discretion? Who decides whether or not it is just to alter or work with certain
organisms? The opinions on such a subject are very diverse, leaving us with no
definitive answer. Many ethical dilemmas like these become evident when working
with and altering living organisms for the sake of scientific inquiry; however
there are many positive benefits to genetically engineering bacteria. For
example, scientist have been able to genetically engineer forms of E. coli so
they secrete proteins that have been found to block HIV from infecting cells of
other living organisms [2 ]. Researchers at Tel Aviv
University have even been able to manipulate bacteria so they light up when
they come in contact with certain pollutants in water [1]. Bacteria
have been genetically modified for many different reasons; many of them have
had an incredibly positive impact on the world around us. As long as the
benefits of bacterial transformation continue to outweigh the risks, their use
will remain extremely important to humanity and beyond.
[1] Blajchman, A. (2009, March 20). Genetically
Engineered Bacteria to Measure Water Quality. Clean Tech News & Views.
Retrieved January 15, 2012, from
http://cleantechnica.com/2009/03/20/genetically-engineered-bacteria-to-measure-water-quality/
[2] Bacteria modified to combat HIV. (2005, November
13). BBC News. Retrieved January 15, 2012, from http://news.bbc.co.uk/2/hi/health/4692905.stm
[3] General Applications of GFP. (n.d.). Green
Fluorescent Protein. Retrieved January 15, 2012, from userpages.umbc.edu/~jili/ench772/application.html
[4] Goodsell, D. (n.d.). Green Fluorescent Protein
(GFP). Protein Data Bank. Retrieved January 15, 2012, from
www.rcsb.org/pdb/101/motm.do?momID=42
[5] Gregory, M. J. (n.d.). Bacterial Transformation
Lab. The Biology Web. Retrieved January 15, 2012, from
faculty.clintoncc.suny.edu/faculty/michael.gregory/default.htm
[6] Hanahan, D. (1983). n.a.. Studies on
transformation of Escherichia coli with plasmids (pp. 166, 557). n.a.:
n.p..
[7] Hanahan, Douglas, Techniques for
transformation of E. coli. In DNA Cloning: A Practical Approach
(Ed. D. M.
Glover), vol. 1. IRL Press, Oxford (1987).
[8] Mulligan, M. E. (n.d.). The Arabinose Operon. Memorial University.
Retrieved January 15, 2012, from www.mun.ca/biochem/courses/3107/Topics/Ara_operon.html
[9] Schleif, Robert, Two positively
regulated systems, ara and mal, In Escherichia coli and Salmonella,
Cellular and Molecular Biology,
Neidhardt. ASM Press, Washington, D.C. (1996).
[10]
ampicillin medical facts.
(n.d.). Drugs.com | Prescription Drug Information, Interactions & Side
Effects. Retrieved January 15, 2012, from http://www.drugs.com/mtm/ampicillin.html
Appendix
Table 1
Table 2
Figure 1
Figure 2
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