Micah the use of plasmids to this end.

Micah Michaud

Final Lab Report

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Life 102 – Lab Section 34

Transformation of Fluorescence and
Ampicillin Resistance via Plasmid Vectors in E. Coli

Introduction:

Genetic
transformation occurs when an organism absorbs foreign genetic material into
its genotype (Sinha and Redfield 2012). There are multiple manners in which
foreign DNA can penetrate the defenses of a cell and be absorbed, in our
experiment we explore the use of plasmids to this end. Probability of
successful transformation via a plasmid can be improved by multiple factors two
of which are temperature and chemical composition of the surrounding solutions (Lorenz
and Wackernagel 1994). An increase of temperature results in increased
permeability of the cells membranes thus increasing chances of the plasmid
passing through the membrane (Hanahan 1983). The extent to which a cell is
capable of absorbing for DNA is called competency. Environmental factors such
as the previously mentioned temperature and chemical environment of a cell can
either increase or decrease competency of the organism. In our experiment we
explore the impacts of these environmental factors on the competency of the
bacteria Escherichia Coli (E. Coli) as well as the process of transformation
that occurs (Williams 2016). Deeper understanding of genetic transformation and
methods in which it can be reliably controlled have significant implications in
the advancement of a multitude of technological and academic fields such as medicine,
synthetic biology, environmental sciences, and energy. Refinement and understanding
of organism specific reactions to transformation of varying traits allows for
more effective and knowledgeable experimentation and application with genetic
transformation at large.

            Targeted competency of E. Coli was evaluated through
attempted genetic transformation of two foreign traits. Green Fluorescent
Protein (GFP) is a protein produced in Aequorea Victoria, a species of
biofluorescent jellyfish. The GFP protein displays fluorescence when the sugar
arabinose is metabolized in its presence (Tsien 1998). Ampicillin is an
antibiotic that is toxic to E. Coli which some bacteria poses a genetic
resistance to (Hanahan 1983). Plasmids containing the genetic material
necessary for the transformation of the aforementioned traits in E. Coli were
introduced to a sample of E. Coli following thermal and chemical treatments
targeted at increasing the competency of the E. Coli samples. After the transformation
the samples were cultured on a series of plates with adequate nutrient and a
varying selection of test solutions. In the final review of the cultured
samples, certain plates should display fluorescence due to a presence of
arabinose and ampicillin resistance while a control plate should show no signs
of growth due to the presence of ampicillin and lack of ampicillin resistance
in the stock E. Coli sample. We hypothesize that a successfully transformed sample
of bacteria will display fluorescence and ampicillin resistance when cultured.
This will be confirmed by the presence of fluorescing colonies cultured on a
plate containing ampicillin and arabinose while a similar plate with non-transformed
E. Coli will fail to grow or survive.

Materials
and Methods:

(a)   Overview

In
our experiment we attempted to demonstrate genetic transformation in E. Coli of
two traits, fluorescence (GFP) and resistance to the antibiotic ampicillin
(AMP). A plasmid (pGLO) containing the genetic information necessary for
genetic transformation of both traits in E. Coli was used as the vector for our
experiment (Weedman 2016). Competency of the E. Coli was increased through the
addition of a transformation solution of CaCl2 and manipulation of
temperature known as Heat Shock (Hanahan 1983), (Weedman 2016). 

(b)   Plates, Tubes, Equipment, Solutions,
and Designations

For
this experiment, four plates were used to culture the final bacteria for final
review. The plates were labeled as follows and contained the corresponding
materials +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. “+pGLO”
indicates a plate with the transformed E. Coli sample, “- pGLO” indicates the
stock (control) E. Coli without having been transformed by the pGLO plasmid, “LB”
indicates presence of Luria Bertani broth (a nutrient broth to facilitate
bacterial growth), “amp” indicates the presence of ampicillin in the agar, and
finally “ara” indicates the presence of arabinose (the necessary sugar for
fluorescence of GFP) (Weedman 2016). Two microcentrifuge test tubes were needed
and labeled as +pGLO and – pGLO, several sterile loops were required for
manipulation of the samples, a centrifuge, hot water bath at 42°C, an ice bath,
micropipetter, and sterile micropipette tips were also required. A plate of non-transformed
E. Coli cultures, a solution of the pGLO plasmids, transformation solution, LB
solution, and the prepared agar plates were the required as well (Weedman
2016).

(c)   Procedure

The
following procedure is recounted from the process performed by our group based
on the procedure outlined in the lab manual (Weedman 2016). Two microcentrifuge
tubes were labeled as +pGLO and -pGLO after which a micropipetter with a
sterile tip was used to transfer 250µL of transformation solution into each of
the two tubes. The two tubes were placed into an ice bath and two sterile loops
were used to transfer an untransformed E. Coli colony into the + and – pGLO
tubes. A new sterile loop was submersed in the pGLO plasmid solution and then
transferred into the transformation and E. Coli solution in the +pGLO tube (Weedman
2016). The tubes both remained on ice for 10 additional minutes during which 4
agar plates were retrieved and labeled as +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO
LB/amp, and -pGLO LB. After the 10-minute cooling period for the
microcentrifuge tubes the tubes were placed into a 42°C hot water bath for 50
seconds and then placed immediately back into the ice bath for an additional 2
minutes. After the 2-minute cooling the tubes were placed in a room temp rack
and a pair of fresh micropipette tips were used to transfer 250µL of LB
solution to the + and – pGLO tubes (Weedman 2016). The tubes were then closed
and incubated for an additional 10 minutes at room temperature after which the
tubes were mixed by flicking them. Another pair of fresh pipette tips were used
to transfer 100µL of the transformation solution into each of their corresponding
+ or – pGLO agar plates. Finally, four sterile loops were used to gently
distribute the solutions throughout the agar plates, the plates were labeled
with group names, and stored upside down in the 37°C incubator until they could
be reviewed a week later (Weedman 2016).

Results:

Four
agar plates were used to culture two control samples and two treatment samples
of E. Coli. The first control plate contained a non-transformed (-pGLO) E. Coli
sample cultured in agarose gel and nutrient broth, the second control plate
contained a non-transformed (-pGLO) E. Coli sample cultured in agarose gel,
nutrient broth, and ampicillin, the first treatment sample (+pGLO) of E. Coli
was cultured in a plate containing agarose gel, nutrient broth, and ampicillin,
the final plate was again the treatment (+pGLO) E. Coli sample but cultured in agarose
gel, nutrient broth, ampicillin, and arabinose. Both the control and
transfected sample were put through the same hot and cold baths, centrifuge
processes, and applied to their respective plates in the same manner (Weedman
2016). Upon review of our cultured transfections it was discovered that a complete
failure of growth occurred in both the control sets and the treatment sets of E.
Coli Image A.
Growth failure of all samples occurred consistently across all of lab section 34
and was not isolated to a single group nor did any observed groups in section 34
have any apparent qualitative or quantitative differences between their samples
to our knowledge. An alternative lab section was successful in their
transfections and provided their samples for review in this experimentImage-B (Apodaca et al. 2017).

Image A:                                                                                             Image B:

                                                                                 (Apodaca
CC, Balbo A, Berman B, and Boehrer J. 2017)

            Review of the successful transfection provided by Apodaca
et al. shows significant growth with consistent
coverage and no discernable concentrated colonies on the first controlImage-B containing the non-transformed E.
Coli and growth media and the second control shows no growthImage-B when the non-transformed E. Coli was
cultured in the presence of ampicillin. The first treatment sample shows reduced
but significant culture growth with 13 small concentrated colonies discernableImage-B when
+pGLO was cultured in growth media with ampicillin. The final treatment culture
done in growth media, ampicillin, and arabinose using +pGLO displays some
culture growth with 3 large concentrated coloniesImage-B (Apodaca et al. 2017). Under a black light only
the final treatment group fluoresced while the LB/amp treatment group showed no
fluorescence nor did either of the control plates (Apodaca et al. 2017). No
fluorescence was observed in any of our groups control or treatment culture plates.

            Discussion:

            Successful transformation of the GFP gene and ampicillin resistance
gene into an E. Coli sample was expected to result in an E. Coli culture
capable of growing in an ampicillin rich environment and fluorescing in the
presence of arabinose. These traits could be correlated to the genetic
transformation by observation of the intended traits when compared to a sample
that was not transformed using the pGLO plasmid under similar developmental
conditions. Our groups experimental results failed to support any hypothesis
due to failure of our samples to grow in either the control or treatment groups.
To produce more meaningful results using our own experimental data we would
require additional trials which were not a viable option due to time constraints.
In lieu of performing additional trials, we were provided with data from a
successful lab section to evaluate the experiment accurately (Apodaca et al.
2017).

Comparison
of both sets of experimental data and procedural information from the lab
manual allow us to deduce possible sources of our experiments failure. Because
the first control plate with -pGLO LB failed to grow as well as the first
control plate with +pGLO LB Amp we can make a few deductions on the assumption that
there was likely a single error that caused the failure. Because other lab
sections succeeded we can safely assume that the error was not one with the
procedure provided by the lab manual which the other sections also followed,
otherwise their results would have been the same (Weedman 2016). Because both
the control and the treatment group failed the error most likely lies within
one of the procedures that were performed on both samples, this precludes the possibility
of failure to transform the treatment group. The remaining sources of error are
a bad or dead stock E. Coli sample, improper incubation, excessive heat or cold
during heat shock, toxic transformation solution, or improper transfer of E.
Coli to the plates. Due to the failure of multiple groups and the relative
simplicity of colony transfer to or from the plates it is unlikely that colony
transfer was the culprit. Improper incubation can be eliminated because
successful groups were incubated in the same manner, because the ice was not
salted the temperature should be the same across all lab sections, and finally to
my knowledge this issue was isolated to our lab section and did not impact the
one that followed it the temperature of the hot water bath would not have been
altered (Weedman 2016). This leaves the most likely causes being a bad stock E.
Coli culture or possibly an issue with the transformation solution which was
added to both the control and treatment groups (Weedman 2016).

Review
of the successful experiment shown in Image B allows us to evaluate our
hypothesis based on the original assumptions that our stock materials were
correct (an assumption we failed to test before performing the experiment) (Apodaca
et al. 2017). Failure of sample growth in the -pGLO + ampicillin control group
confirms that the E. Coli is unable to survive in the presence of ampicillin which
in combination with successful group in the +pGLO + ampicillin treatment group
confirms that the pGLO trait does provide ampicillin resistance to the E. Coli
if transformation is successful (Hanahan 1983). Lack of luminescence in all but
the final treatment group containing arabinose and ampicillin confirms that the
luminescence trait is present in combination with ampicillin resistance as well
the necessity for arabinose for the trait to present. Our experiment does not
allow us to make any definitive statements as to whether arabinose in the
presence of the non-transformed E. Coli would behave differently due to the
lack of a control sample containing arabinose (Tsien 1998).

Due
to the relative success of the experiment in combination with the natural
competency of E. Coli it is likely that the assumption that the application of
heat shock and transformation solution increase competency is accurate (Sinha and
Redfield 2012). Addition of CaCl2 as a transformation solution increases
the positive ion concentration in the solution and attracts both the DNA within
the cell and the plasmids that have been added (Weedman 2016). Heat shock increases
competency by increasing attraction of the polar molecules while cold and causing
destabilization of the phospholipid bilayer of the cell membrane that is
separating the DNA from the plasmid (Hanahan 1983). The combination of these
two effects causes the plasmid to more easily breach the cell membrane and
transform the DNA. Our experiments did not explicitly test these two theories,
however the natural success rate of transformation via plasmid in E. Coli is
fairly low implying that our success was likely aided by this process (Sinha
and Redfield 2012). To determine ideal processes for transformation of E. Coli
using a plasmid regarding heat shock and ion concentration additional
experimentation varying the temperatures, types of ions, and concentration of
ions in solution would be needed.

Conclusions:

Although
our individual experiment failed due to ultimately unknown          complicationsImage-A we were able to analyze our
hypothesis using data provided by a trial performed by a different lab section
(Apodaca et al 2017). Failure of -pGLO E. Coli to grow on the control plate
containing ampicillinImage-B
indicates inability of E. Coli to survive in an environment with a significant concentration
of ampicillin (confirmed by the -amp control plate) while its failure to
fluoresce indicates that, at minimum, it requires the presence of arabinose to
fluoresce. Survival without presenting fluorescence in the first treatment
plate of +pGLO with ampicillin and no arabinose while the final plate
containing +pGLO, arabinose, and ampicillin survived and fluoresced indicates
that arabinose is a necessity for fluorescence of ampicillin (Apodaca et al
2017). Culture colony and size appears to be significantly impacted by a
combination of factors although the means through which do not appear to be
clear. Additional trials would be required to further isolate whether reduced
colony count with increased size occurred in the presence of arabinoseImage-B (Apodaca et al 2017). Our initial
hypothesis holds true based on the collected data; however, it does lack
specificity with regard to the role of pGLO as the source of the fluorescence.
In order to completely confirm the intent of our hypothesis an additional control
with non-transformed E. Coli needs to be cultured on a plate with agarose, LB,
and arabinose so that we can determine if there is any natural fluorescent
property present in either the E. Coli or Arabinose.

 

 

 

 

Literature
Cited:

Sinha S, Redfield RJ.
2012. Natural DNA Uptake by Escherichia Coli. PLOS ONE e35620.

Tsien RY. 1998. The Green
Fluorescent Protein. Annual Review of Biochemistry. 67:1, 509-544.

Hanahan D. 1983. Studies
on transformation of Escherichia Coli with Plasmids. Journal of Molecular
Biology. 166:4, 557-580.

Lorenz MG, Wackernagel W.
1994. Bacterial Gene Transfer by natural Genetic Transformation in the
Environment. Microbiological Reviews. 58:3, 563-602.

Williams AB. 2016.
Section II: Genome Instability in Bacteria and Archaea. In: Kovalchuk I,
Kovalchuk O, editors. Genome Stability: From Virus to Human Application.
Boston: Academic Press; 978-0-12-803309-8. p 51-85.

Weedman D. 2016. Life
102: Attributes of Living Systems Lab Manual. 8th ed. Minneapolis:
Bluedoor, LLC. p 67-160, 179-183.

Apodaca et al. 2017 Genetic
Transformation of E. Coli Experiment: Culture Plate Image. Fort Collin: Colorado
State University.

 

Acknowledgements:

Special thanks to Apodaca
CC, Balbo A, Berman B, and Boehrer J. for providing their successful
experimental samples for use in this report.

 

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