Positive And Negative Controls In Bacterial Transformation Experiments

Identify And Explain The Difference Between Positive And...
1
What is the purpose of including Controls #1 and #2?
What does each of these control for?
Which might be considered a positive control? A negative control? Why?
Controls are the standards. They can show what the experiment would normally happen or not
happen. By comparing the controls with the experimental trials, we can tell whether the independent
variables have an effect on the cells (and sometimes it can indicate if there is contamination
throughout the experiment).
Control #1 had all three components of the transforming extracts –– DNA, RNA and protein ––
without enzyme. It controlled the use of enzymes and made sure that the all three components of the
transforming extracts are accessible to the competent cells. Control #1 assessed ... Show more
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Control #1 would have a known response, where the competent cells transformed to be ampicillin–
resistant. In other words, Control #1 should grown on both LB plate and LB+antibiotic plate.
Control #1 would provide a good positive response for comparison with the other unknown
responses (growth or no growth) of the treatments by different enzymes that broke down each of the
three components of the transforming extracts. Therefore, it could be seen whether a component
broken down by enzyme was essential for the transformation (transfer and inheritance). Control #2
was the negative control because no response was expected, and Control #2 should not have a
response. In other words, Control #2 should grow on the LB plate but not on the LB+antibiotic
plate. If it did have a response, there must have been something wrong with the experiment (eg.
contamination of the competent cells).
2
Why are all the transformations being plated on LB+antibiotic? On LB alone?
LB plates could prove that E. coli was alive and active after each treatments. It served as
controls/standards for comparison with LB+antibiotic plates. LB+antibiotic plates could prove
which E. coli was ampicillin–resistant.
3
What are your expectations for each of the 10 transformation
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Ampicillin In Bacteriophage Experiment
We have shown that bacteriophage lambda can be cloned and amplified, ligated and transformed,
and inserted successfully into bacterial cloning vector pUC18. The results from the experiments
above can be used not only to clone known genes, but also to discover unknown genes that have
potential in medicine and gene therapy.
Control 4 is a positive control, and is set as an example of whether or not the digestion of pUC18
had been successful. Controls 5 and 6 illustrates whether the ligation reactions were successful
during the experiment. Control 7 is a negative control, and illustrates whether the ampicillin in the
agar solution was working.
Reactions 1–3 were set up to locate the optimum ratio of bacteriophage insert DNA to create the
most ... Show more content on Helpwriting.net ...
coli cells. Removal of all or part of the non–essential region, between positions 20–35 using
restriction enzyme HindIII decreases the size of the resulting molecule by up to 15 kb (Brown,
2006). Since during this experiment that bacteriophage DNA molecule wasn't modified not all
fragments were recovered equally, therefore the transformation efficiency is decreased.
The experiment completed needs to be improved as according to previous studies, figure 1, has been
performed incorrectly either through operator error in multiple stages of the experiment, or an
unknown error. Lane 5 was incorrect as according to figure 3, obtained from group 32 results, two
distinct bars are meant to be apparent at 4,500 bp and 1000 bp. The data could be improved by
performing the experiment a second time to ensure that all results are correct and no human error
has occurred.
Another improvement that can aid in the accuracy of the experiment would be to use combinational
cloning, a less complex and involved technique for efficient cloning of different vectors with various
clone sites (Zhang and Tandon, 2013). The technique uses originally existing clone sites from
circular plasmids to prepare the inserts to achieve maximal correct digestion of the inserts (Zhang
and Tandon,

Lb-Pglo Dish Lab Report
Results: There of the dishes turned out as expected in this experiment. Our group expected there to
be growth in the LB –pGLO dish as the bacteria were not exposed to the antibiotic ampicillin.
Furthermore, our group also expected to see inhibited bacterial growth in the LB/amp +pGLO dish
as there was ampicillin in the dish, but some of the bacteria were immune as they possessed
immunity to the ampicillin. Moreover, our group expected that there would be no bacterial growth in
the LB/amp –pGLO dish, as the bacteria were exposed to ampicillin and were not immune.
However, the final dish, LB/amp/arbo +pGLO, did not turn out as expected. While it was expected
to allow for inhibited bacterial growth and the bacteria to become florescent, ... Show more content
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This is proven by the survival of the bacteria in the LB/amp +pGLO dish, as the same bacteria that
lacked the modification were killed by the ampicillin. However, we were unsuccessful in genetically
transforming bacteria to be fluorescent. This is because in the LB/amp/arbo +pGLO dish, we were
unable to locate any bacteria.
An organism's traits are often caused by a combination of its genes and the environment it lives in.
Think about the green color you saw in the genetically transformed bacteria:
What two factors must be present in the bacteria's environment for you to see the green color?
For the bacteria to become fluorescent, the environment must contain arabinose and the bacteria
must have the GFP gene. Furthermore, the bacteria must have a nutrient supply in order to thrive. In
this experiment, this was provided by the LB nutrient.
Explain how each of the two environmental factors you listed above are causing the genetically
transformed bacteria to turn green.
The GFP gene allows for the bacteria to have the information to produce the fluorescent color.
However, this is not enabled by default. In order for it to be activated, it must be induced by the
arabinose. Once arabinose is introduced, the protein can be

The Domestication of Animals Was the Percursor to Genetic...
The domestication of animals 30,000 years ago was the precursor to Genetic Engineering. Starting
with the dog we have since been able to introduce desirable traits in all organisms. With the
discovery of plasmids in the late 60's we have been able to take genetic engineering even further.
Plasmids are small circular DNA molecules used to amplify and replicate a gene of interest. These
minute molecules have the ability to replicate with the chromosome or independently, allowing them
to have up to a 100 copies in one cell. Plasmids are important because of their characteristics to
transfer genes that occur naturally within them or acting as a vector to introduce foreign DNA into a
host cell. With the use of plasmids genetic engineers are able to use bacterial transformation to make
medicines. Bacterial transformation is the process in which a bacterial cell takes up foreign DNA
and incorporates this DNA into its own. With the use bacterial transformation this part of Genetic
Engineering has become the most important and widely used technique, creating life saving
antibiotics and medicines. E.coli is generally used in these procedures because of its ability to adapt
and grow exponentially. Genetic Engineers can then use large bacterial colonies, Ampicillin, and X–
gal to indicate if b–glactosidase is present along with identifying the recombinant and non–
recombinant colonies. The transformed bacteria that contains ampicillin will spread and survive
either turning white

Lab Report Plasmid Pglo
Bacterial Transformation Lab Report Backround:
The plasmid pGLO contains an antibiotic–resistance gene, ampR, and the GFP gene is regulated by
the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E.
coli cells contain the ampicillin–resistance gene, the cells can survive exposure to ampicillin since
the ampicillin–resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed
E. coli cells containing ampicillin–resistance plasmids can easily be selected simply growing the
bacteria in the presence of ampicillin–only the transformed cells survive. The ara control region
regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and ...
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6. Incubate both microcentrifuge tubes on ice for fifteen minutes.
7. Take both tubes out of ice and immediately place in incubator at 42٥C for 90 seconds.
8. After place both tubes back in the ice for two minutes.
9. Add 200uL Luria Recovery Broth to both microcentrifuge tubes.
10. Let both the tubes rest at room temperature for 10 minutes.
11. During the 10 minutes, get the LB agar and LB+AMP agar plates ready. Mark your plates with
the transformation tube mixture to use (+ or –), the lab group names, and the date on the top of the
dishes. 12. Add 100ul of the pGLO transformation cell mixture to the center of the agar surface of
the corresponding LB agar and LB+AMP plates.
13. Use a sterile plastic loop to distribute the cell suspension evenly on the plate by "skating" the
loop back and forth across the LB agar plate several times.
14. Use the same loop and technique to spread the same cell suspension (+) on the LB+AMP agar
plates. Dispose of the sterile loop in a beaker of germicide.
15. Repeat the procedure by spreading the (–) transformation cell mixture to each of the (–) labeled
LB and LB+AMP plates. Be sure to use a fresh plastic loop for the 'None' transformation mix.
16. Stack your group's set of plates on top of one another and tape them together. The plates should
be left upright position to allow the cell suspension to be absorbed by the agar.
17. Place the plates in an inverted position (agar side on top) in a 37٥C

Ampicillin Resistance Lab Report
Discussion
As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an
ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the
LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the
plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no
ampicillin in its environment and no plasmid treated E. coli served as a positive control for this
experiment because it demonstrated how the E. coli would colonize and grow in a normal setting.
The cells in the positive control plate grew into lawn colonies because they were not placed into a
selective environment or transformed, so they had no need to acquire ampicillin resistance. Two
plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the
lux plasmid but were placed in an ampicillin free environment. These two colonies grew ... Show
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In order to find transformation efficiency, the total number of colonies on the plate is divided by the
total amount (µg) of DNA spread on the plate. All the factors that must be taken into account while
finding the transformation efficiency include; the total amount (µg) of plasmid DNA used, the total
volume (µl) of cell suspension prepared, the fraction of DNA spread on the plate and finally the total
amount (µg) of DNA present on the plate. In the LB/AMPC plate the transformation efficiency was
8.2 x 103 colonies per µg of plasmid DNA while the LB/AMPlux plate the transformation efficiency
was 1.07 x 104 colonies per µg of plasmid DNA. The transformation efficiency for the LBC and
LBlux plates were not taken into account, even though they too transformed because they were not
in a restrictive environment where they needed to express their ampicillin resistance genes in order
to

Genetic Engineering And The Human Genome Project
In 1990, the science world began a vigorous new exploration of human DNA– the Human Genome
Project. The goal of this project was to map out all the human genes (An Overview of, 2015), which
ultimately led to a deeper understanding of all genes, not just a human's. This deeper understanding
also helped scientists to progress further in the technology of recombinant DNA. Recombinant DNA
is when DNA from different cells is spliced together, creating a new strand (Kuure–Kinsey, 2000).
Recombinant DNA is often used to genetically change a cell, which is known as genetic
engineering. Genetic engineering can be used to prevent and alleviate symptoms of various diseases,
by pinpointing and fixing the gene that causes them. It enables organisms to ... Show more content
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Instead of testing genetic engineering directly on a human, scientists perform tests on other living
things– like the Escherichia coli bacteria used in the pGLO lab. The purpose of the scientists
performing the pGLO lab was to see if they were able to alter the DNA of a bacteria, E. coli, causing
the bacteria to glow, if the insertion of the glow gene, which originated in a jellyfish, was successful.
Four groups of bacteria were experimented with. Two of these groups were control groups; the
bacteria with no DNA alteration on an agar plate containing nutrient broth, and the bacteria with the
pGLO gene added on an agar plate with both Ampicillin and nutrient broth added to it. Each of these
plates acted as a baseline for comparison for the other two plates, which were the experimental
plates. The experimental groups were the groups consisting of unaltered bacteria on an agar plate
containing Ampicillin and nutrient broth, and the altered (pGLO gene added) bacteria on an agar
plate containing the nutrient broth, Ampicillin, and Arabinose sugar. Along with control groups and
experimental groups, every experiment has both independent and dependent variables. In the pGLO
experiment, the independent variables were the Ampicillin, Arabinose sugar, and the nutrient broth,
all of which were added to the agar plate, as well as the pGLO gene being added to the bacteria's
DNA. These

Antibiotic Resistance Of The Gram Negative Bacterial...
Antibiotic Resistance of S. marcescens
Yesong Liu
Comparison of Antibiotic Resistance of Serratia marcescens to Streptomycin and Ampicillin
Abstract
The purpose of this study and experiment was to analyze and compare the relative antibiotic
resistance of the gram–negative bacterial species Serratia marcescens to the antimicrobial species
streptomycin and ampicillin. Preceding researches have shown that streptomycin has a stronger
effect on the bacteria species than the ampicillin. In this experiment, the disc–diffusion method has
been introduced and the zone of inhibition was measure in millimeters around the disc saturated
with the same molar concentration of the two antibiotics– streptomycin and ampicillin. The
hypothesis is that if the colonies of S. marcescens were treated separately with streptomycin and
ampicillin with all other factors remaining the same and controlled, the S. marcescens would display
a stronger resistance toward the ampicillin than to streptomycin. The results from the experiment
supports the hypothesis that S. marcescens is more resistant to ampicillin than to streptomycin.
Introduction
Serratia marcescens is a species of Gram–negative bacteria. S. marcescens is considered as a motile
organism and expected to be found in soil, dirt or sterile locations. Potential infections would be
caused by would sites, respiratory tract and urinary tract. Its red pigmentation makes it recognizable
in this experiment. In this study, it will be plated to grow

Serratia Marcescens Susceptibility Of The Antibiotics...
Serratia marcescens Susceptibility to the Antibiotics Streptomycin and Ampicillin
Rileigh M. Jones
Biological Sciences Student, Cornell University, Ithaca, NY–14853–2401 e–mail:
rj246@cornell.edu
Abstract–The gram–negative bacteria Serratia marcescens has gained attention in recent years for its
tendency to cause nosocomial infections in humans, as well as its development of antibiotic
resistance. Antibiotic resistance in a bacterium that is harmful to humans can be concerning as it can
result in infections that are difficult to treat. In order to find out more about the growing antibiotic
resistance of S. marcescens, this experiment used the disc diffusion method to test the susceptibility
of S. marcescens to two varieties of antibiotics that were known to have success against some gram–
negative bacterium: streptomycin and ampicillin. These antibiotics were, respectively, an
aminoglycoside and a beta–lactam. The experiment tested which of the two that S. marcescens had
developed more of a resistance to. The zones of inhibition of the discs were significantly larger for
discs treated with streptomycin compared to discs treated with ampicillin. This led to the conclusion
that S. marcescens is less resistant to streptomycin than to ampicillin.
Key Words: Serratia marcescems, antibiotic resistance, streptomycin, ampicillin
INTRODUCTION
While the bacteria S. marcescens was considered to be harmless in the past, it has been identified in
recent years as a cause of serious

Genetic Transformation Lab
Genetic transformation occurs when an organism is modified by the introduction of new genetic
information which is then incorporated into the organism's genome. In this lab, engineered proteins
containing DNA to absorb blue light and emit green light were incorporated into E.coli bacteria. The
main purpose of this lab was to transform E. coli by forcing it to uptake plasmid containing an
ampicillin resistance gene and a green fluorescent protein (GFP) using complementation (adding
antibiotic resistance in addition to weed out non–transformed cells). To see the effects of this
plasmid on cells, bacteria treated with the plasmid were grown 3 different growth mediums: agar,
agar/ampicillin(+), agar/ ampicillin(+)/PTG(+). With the plates prepared, ... Show more content on
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The transformation efficiency is the efficiency by which cells can take up extracellular DNA and
express genes encoded by it. The results from this lab show that this efficiency was 0. There was no
ramification of findings for this lab because the expected results were not obtained. If there were
successful outcomes, more research on gene insertion in bacteria for insulin production and cancer
research could have been further built upon. Since the transformation efficiency calculation was 0,
there were many errors that have arisen. The main problem was there was no control growth, which
means there was a bacterial problem with the culture. Another error could have been problems with
the broth and CaCl2 solutions. With regards to the GFP protein and its ability to be tracked using
UV light, is there another protein out there that scientists and doctors can track without using a UV
light,

Green Color
If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic
ampicillin, then what might be inferred about the other genes on the plasmid that you used in your
transformation procedure?
The plasmid can be inferred to have the gene resistant to the antibiotic; thus, giving the transformed
cells the ability to live while in the presence of ampicillin.
From the results that you obtained, how could you prove that the changes that occurred were due to
the procedure that you performed? Look again at your four plates. Do you observe some E. coli
growing on the LB plate that does not contain ampicillin or arabinose?
One could prove that the changes that occurred were due to the procedure performed by comparing
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When adding the plasmid DNA to the +pGLO tube, the scientists tried to use the loop to get 10μ to
get a film of plasmid across the circle; however, they could not. This could have affected the
resistance to ampicillin and the visible green glow of the bacteria. Some contamination that might
have occur is when the scientist forgot to close the lid to one of the plates or did not close the bag
containing the sterile loops, which could have resulted in bring other bacteria into the

A Summary On ' Pglo Pig Slurry '
pGLO Pig Slurry
Ana Chiman
Frank Alfano
L13
11/19/15
Abstract pGLO is a plasmid that contains several genes, araC, gfp,bla, and an ori of replication. E.
coli was artificially induced that became a competent bacteria when it took the pGLO DNA, so it
had the ability to have ampicillin resistance and fluoresced when arabinose was present. Two tubes
with E. coli were labeled to differentiate which tube the pGLO was added to, then through several
steps the bacteria was induced to intake the pGLO DNA. At the end, each tube was inoculated on to
three different plates that contained different substances and they were incubated then observed. The
results showed only one E. coli culture had growth and fluoresced which was the pGLO+ E. coli
that was grown on the plate with LB, amp +arab, there were only two E. coli cultures that did not
grow because the pGLO– E. coli did not have the ampicillin resistance to grow in ampicillin
conditions, and the rest of the culture plates showed growth. pGLO could be used in food safety
experiments done by the food safety department that helped to identify Salmonella and Yersinia
enterocolitica in pig slurry, so they were able to calculate how long these bacteria lived in certain
conditions before disinfectants were used.
Introduction Transformation is a process when a bacteria cell was able to be incorporated into a
sequence of DNA from the environment, this type of bacteria is called a competent

Different Methods, Bacteria, And Development Transduction,...
Through three different methods, bacteria can obtain new genetic material: conjugation, transduction
and transformation Transduction is when a bacteriophage inserts it 's DNA into the host bacteria
cell, while conjugation is when the bacteria shares DNA through direct contact, eg. horizontal gene
transfer. The third method, transformation, is the intake of external DNA into the cell (Chen and
Dubnau, 2004). In order for bacteria to be able to take in extraneous DNA, they must first be in a
state of competent (Hanahan, 1983). There are two types of transformation cells in transformation:
natural competence and artificial competence. Natural transformation can occur in both gram
negative and gram positive bacteria, but have different pathways to take in the new DNA. There are
several ways to induce competence onto some bacteria cells, that are not naturally competence.
Cells can be treated to heat shock, starved of nutrients, the use of electroporation or keep in ionic
solutions to induce competence by increasing the permeability of the membrane (Van der Rest et al.,
1999). Plasmids are circular double–stranded DNA found in bacteria that can encode for basic
necessary functions (Couturier, et al., 1988). Ampicillin (6[D(–)–a–aminophenylacetamido]
penicillanic acid) is an antibiotic that is a derivative of penicillin and is can work against both gram–
negative and gram–positive bacteria (Acred et al. 1962)
The experiment insisted of insertion of plasmids that

The Transformation Of E. Coli
Jack Dildabanian
Ms. Buckley
Genetics
11/6/15
The Transformation of E. coli using the plasmid GFP
Purpose: The purpose of this lab is to observe bacterial growth under various conditions including
the transformation of bacteria; to understand how the process of transformation occurs.
Background Information: Transformation is the "process by which the genetic material carried by an
individual cell is altered by the incorporation of foreign (exogenous) DNA into its genome"
(MedicineNet.com, "Definition of Genetic transformation"). Transformation in bacterial cells occurs
when the cell incorporates DNA into its genetic material. Bacteria cells that have the ability to take
up DNA are called "competent." In a lab setting, this is encouraged by placing the mixtures of
transformation solution and plasmid DNA on ice, then rapidly transferring them to a hot water bath
for about fifty seconds, and then placing them back on ice again. This procedure is called heat shock
and increases the permeability of the cell membrane to DNA. The agent which the new genetic
material is incorporated into is the bacterial plasmid. A plasmid is a circular deoxyribonucleic acid
(DNA) molecule that replicates independently of the bacterial chromosome and often permits a
bacteria to gain resistance to an antibiotic. Recombinant plasmids are those which have DNA from
two or more sources incorporated into a single plasmid. To make recombinant plasmids, two
different plasmids are cut with the same

If Genetic Transformation Has The Meaning Of Change Caused...
If Genetic transformation has the meaning of "change caused by genes" and involves the placing of
a gene into an life form in order to modify the organisms characteristic; the progression of placing
genes from one life form to a different is used to assist of a plasmid and the pGLO plasmid codes
the gene used for GFP as well as the gene for resistance to ampicillin. It is used to manage the
expression of the fluorescent protein; hence, the GFP gene is able to be switched on by adding the
sugar arabinose to nutrient medium of the cell, then the bacteria will be able to glow a bright green
underneath UV light when arabinose is within the nutrient agar medium. Hence, then when one
micro test tube +pGLO and –pGLO are labeled and placed into a ... Show more content on
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III. Methods
See lab sheets for both electrophoresis and pGLO
IV. Data Bio Rad pGLO
1. Lesson 2 Review questions #1–4 pg.42
i. The plate that I would expect to find bacteria in a large amount that is similar to the original non–
transformed E.Coli colonies is plate that includes –pGLO and LB because this plate is the control
for the experiment. The control plates (–pGLO/LB) will have majority of the bacteria like the
original untransformed E. coli because the bacteria removed from the starter plate didn't contain
plasmids. Therefore, the non–transformed E.Coli colonies will be all over the plate forming a lawn
of bacteria. The pGLO demonstrates that there are no plasmids present living on the plate. Hence,
every cell grows because there is no present on the antibiotic on the plate.
ii. The genetically transformed bacterial cells would most likely be on the plates that have +pGLO
LB/amp or +pGLO LB/amp/ara. The plate with LB/amp, the cells with

How Does Pglo And Ampicillin Affect The Growth Of Bacteria
How pGlo and Ampicillin Affect the Growth of Bacteria
Transformation is any change in an organism that alters its general character. It means "change
caused by gene." (biology–online.org) Genetic engineering is the process of manually adding new
DNA to an organism. The main goal of genetic engineering is to add one or more new traits that the
organism does not already have. ( ) There are many benefits of genetic engineering. With genetic
engineering many illnesses and diseases can easily be prevented by isolating the gene that causes
them. Also, genetic engineering has the ability to increase genetic diversity and pinpoint desirable
traits of certain organisms. There are also various disadvantages of genetic engineering. For
example, the process of genetic engineering is a long process that is risky and tricky. You need a
variety of information in order to attempt the process. There is also debate about genetic
engineering. Some people believe that it messes with the moral issues in religion.( ) ... Show more
It was also conducted to learn about the process of moving genes from one organism to another with
the help of a plasmid. The control group was the –pGlo with LB and ampicillin antibiotic, and the –
pGlo with LB. The experimental group was the +pGlo with LB and ampicillin antibiotic, and the
+pGlo with LB, ampicillin antibiotic, and arabinose. The dependent variable was the bacteria. It had
the ability to change. The independent variable is the ampicillin antibiotic and pGlo. They stand
alone and are not changed by other factors. The organism used was the bacteria GIVE TYPE OF
BAC. and HOW WILL THE BACTERIA BE

Discussion Test Lab Report
Discussion
The expected result from this experiment, was that the plates containing plasmid DNA, GFP, and
IPTG would grow and exhibit a florescent glow. All the plates containing plasmid DNA would grow
and those that contained no plasmid would not grow. The plasmid DNA is able to be absorbed only
if the bacteria is competent, which is a result of the heat shock (Bacteria Transformation and
Selection, 2017). The head shock opens the pores of the membrane allowing the passage of plasma.
Super coiled plasma is easier to be absorbed into the bacteria versus linear plasma (Bacteria
Transformation and Selection, 2017). These expected results were correct for the plates containing
the plasmid DNA. The results deviated for the plates that contained no ampicillin and no DNA
plasmid. These ... Show more content on Helpwriting.net ...
This was seen in both trial A and B, where the plates exhibited no growth (Table 1, Table 2). The
lack of plasmid in the presence of the ampicillin, makes the growth of bacteria impossible because
the ampicillin destroyed the E. coli cells by breaking down their cell walls (How do Antibiotics...,
2006). The bacteria had no added substance which would make them resistant to the ampicillin
(Edvotek, 2014). Since no growth occurred on either plate, the transformation efficiency was unable
to be evaluated (Table 1, table 2). This plate was used as a control to show that without ampicillin
bacteria would not be able to grow without plasmid DNA. This plate can be used to specifically
compare against the plate that contained ampicillin and plasmid DNA. By comparing these two
plates, one can see the growth that was able to occur in the plates that contained the plasmid DNA
versus the plate that contained no plasmid DNA. In trial A, these two plates cannot be compared
because both plates lacked growth (Table 1). In trial B they can be compared because the plate
containing the plasmid DNA was able to grow (Table

Using The Method Of Heat Shock
Introduction:
Genetic Transformation is the process by which a segment of DNA of a specific organism is
implanted into another organism that reads the foreign organisms DNA and presents the phenotype
for the selected DNA segment. The genetic transformation can occur using three different methods,
but in the case of the experiment conducted and described below, was performed using the method
of heat shock. In the experiment, heat shock was used to genetically transform a piece of DNA from
a jellyfish into a sample of E. coli bacteria to observe if the transformation would occur. The
components necessary for the transformation were: pGLO, a plasmid acting as a vector for the
transformation and contained the DNA segment extracted from jellyfish that encodes for the
production of a protein that glows (GFP); Luria Bertani broth, the culture for the transformation;
Ampicillin Resistant gene, to sort cells so only the cells having absorbed plasmid will survive;
Arabinose, a sugar to aid in the growth of bacteria. The genetic transformation was tested using four
samples, each having a different environment. The first sample's environment consisted of +pGLO,
the Luria Bertani broth, and a gene resistant to Ampicillin. The second sample's environment
consisted of +pGLO, the Luria Bertani broth, a gene resistant to Ampicillin, and arabinose. The third
sample's environment consisted of –pGLO, the Luria Bertani broth, and a gene resistant to
Ampicillin. The fourth sample's environment

Scientific Experiment: Bacterial Transformation
Title:
Research Question:
Can modifying the genetic material of bacteria create new characteristics through gene transfer?
Introduction:
Prokaryotic cells have no nucleus and no internal membrane. Because of this, they can be classified
as bacteria. Prokaryotic cells are similar in structure to Eukaryotic cells, however the DNA floats in
a nucleoid. Transformed bacteria consist of a recombinant plasmid and a host cell or bacteria. If the
plasmid in the bacteria has a gene that is useful it can be very beneficial to the bacteria. Scientists
can combine new DNA that they want replicated with the plasmid, so they can have large amounts
of it. The bacteria may pass on the plasmid onto another bacteria, creating many lawns or colonies,
which ... Show more content on Helpwriting.net ...
3.Place both tubes on ice for 15 minutes.
4.Add isolated colonies of E.Coli to the +plasmid tube using an inoculating loop. Make sure
colonies are at the bottom.
5.Repeat step 4, but with the –plasmid tube.
6.Return both tubes to ice.
7. Using a new inoculating loop, add one loop–ful of plasmid DNA to the +plasmid tube, mix it in
the tube, and return it to ice for 15 minutes.
8. During the incubation, label 6 agar plates the following: a. +Plasmid b. –Plasmid c.
+Plasmid/ampicillin d. –Plasmid/ampicillin e. +Plasmid/ampicillin/x–gal d. –Plasmid/ampicillin/x–
gal
9. After the incubation, heat shock the cells. Do this by removing the tubes directly from the ice into
42degree Celsius water bath for 90 seconds.
10. Return both tubes to the ice for approximately 1 minute.
11.Using a sterile pipette, add 250 µL of Luria Broth to each tube and mix by tapping the tubes.
Leave the tubes on a tube rack at room temperature for 10 minutes.
12.Remove 100 µL of cells from the tube and spread them on the plates with their corresponding
plasmid. (i.e. tubes with +plasmid go to the +plasmid agar plates and vice versa.)
13. Let the plates rest for several minutes to allow the cell suspensions to become absorbed into the
agar.
14. Use a sterile pipette to add 100 µL of cell suspension from the +plasmid to each appropriate
plate, and spread.
15.Seal the plates with tape, and incubate upside down for 24–36 hours at 37 degrees Celsius.

Predictions:
I predict that the plates

How Escherichia Coli To Form A Recombinant Plasmid
Transformation of Escherichia coli to Form a Recombinant Plasmid Containing Genes for both
Ampicillin and Kanamycin Resistance
By Valerie Weeks
Lab Partner: Rachel Fahs
Genetics
Section 71
Dr. Tarun
April 8th, 2016
Abstract
The objective of this experiment was to transform E.coli into having genes resistant for ampicillin
and kanamycin by using recombinant plasmids. The three steps of the experiment include ligation,
transformation, and growth on media. Restriction enzymes BamHI and HindIII splice the DNA. The
recombinant plasmid is formed and combined with E.coli. Four experimental plates and four control
plates were incubated at 37 degrees Celsius for 24 hours, then examined for growth. Lawns of E.coli
grew on the LB plates. The ... Show more content on Helpwriting.net ...
One plate was labeled LB/pAMP+kan "+", one was labeled LB/pAMP+kan "–", one was labeled
LB+, and one was labeled LB–. 100 microliters of the cell suspension from the –pAMP/KAN tube
was put on the LB/amp+kan plate using a sterile transfer pipette. Another 100 microliters was added
to the LB– plate. The cells were spread evenly over the surface of the plates using a sterile metal
spreader. The spreader was sterilized by dipping it in ethanol and flaming it shortly with a Bunsen
burner. When the spreader cooled, the cells were evenly distributed on the plate. Using another
sterile transfer pipette, 100 microliters of cell suspension from the +pAMP/KAN tube was put on the
+LB/amp+kan plate, and another 100 microliters on the LB+ plate. The cells were spread evenly on
the plate using the metal spreader previously described. The plates were left to sit for about 10
minutes before being sealed and incubated at 37 degrees Celsius for 24 hours. After 24 hours, the
plates were removed from the incubator, and then examined for growth. The number of colonies on
each plate were recorded.
Results
Transformed Cells Plate # Plate Type # of colonies
+pAMP/KAN 1 LB/amp+kan 360
+pAMP/KAN 2 LB Lawn
–pAMP/KAN 3 LB/amp+kan 0
–pAMP/KAN 4 LB

Ampicillin On Bacteria With Plasmid
The Effect of Ampicillin on Bacteria Treated with Plasmid
Abstract
This laboratory explored the effects plasmid has on the growth of bacteria in the presence of an
antibiotic. Four plates were tested: Luria agar +amp –plasmid, Luria agar +amp +plasmid, Luria
agar –amp –plasmid, and Luria agar –amp +plasmid. Only two of the plates were treated with
plasmid in order to determine the effect it had on the growth of bacteria. The bacteria treated with
plasmid was able to grow in the presence of an antibiotic while the bacteria not treated with the
plasmid died due to the lack of resistance to the antibiotic.
Introduction
Transformation is the process in bacteria and yeast in which pieces of DNA from the environment
are taken up by the cells and ... Show more content on Helpwriting.net ...
For example, we may have insufficiently mixed the solutions of DNA after adding the plasmid to the
solution. If the plasmid was not mixed in fully, some bacteria may not have been exposed to it,
therefore dieting in the presence of ampicillin, which may have caused minor errors in our results.
Also, not returning the mixtures to ice quickly enough after doing the heat shock may have caused
some bacteria to not fully take in the plasmid. The heat shock is what allows for the bacteria to take
in the new piece of DNA because the cell membrane opens up allowing for the plasmid to enter. In
order to keep the new DNA inside the bacteria, it was crucial to quickly place the bacteria back on
ice following the heat shock because this allowed for the cell membrane to close back up and seal
the plasmid inside. In addition, while spreading the liquid on the Luria agar plate, I accidentally
pushed too hard and broke through the Luria agar. This caused us to get a new plate and redo the
past steps for that plate. Because of this, the timing wasn't kept constant for all of the bacteria, which
could have affected the end results since the bacteria absorbed the liquid for various amounts of time
before placing them in the

Bacterial Cell Lab Report
Transformation of cells was done by inserting the exegonous genetic material from different source
(environment). Then, it was inserted into the host. This process will make the host cell transformed
into competent cell. The exegonous genetic material is particular genetic material that express
important protein or resistancy towards particular things. For example, in this pratical the E.coli was
transformed into competent cell by inserting plasmid that can develop resistance toward ampicillin.
Normally E.coli cannot live on the media that containing ampicillin. By referring to the genetic
expression of cell, the genetic material of bacteria, which is plasmid will express the protein and
develop resistancy. This is what called transformation of competent cell.
Plasmid is circular genetic material that contain inside bacterial cell.(Gerald Karp. 2009.)
Transforming competent cell uses bacterial cell because the cells is very useful and versatile. They
can carry out ... Show more content on Helpwriting.net ...
By looking to this characteristics, bacterial cells can undergoes evolutionary changes that make it
become more resistance toward the things that kill the cells. For example, in this practical the E.coli
was inserted with plasmid that have been cut and contained ampicillin resistancy genetic material.
After the expression of the plasmid, the E.coli will be granted the resistance of ampicillin.
Plasmid that are inserted into the E.coli contain amp resistance that are engineered earlier. The
plasmid are called pUC18. This plasmid are artificial that engineered to contain ampicillin
resistance, gene and the promoter for the enzyme lacZ. The lacZ gene contain restriction enzyme
cutting site and polylinker region. The cutting of plasmid of restriction enzyme that are same by
foreign DNA can be ligase together at the cutting

Bacteria Pathogens
Introduction Bacteria pathogens have become increasingly resistant to antibiotics. (Vuotto et al.
2014) The purpose of this experiment is to understand how bacteria are genetically modified in
order to better understand the changes going on naturally in such bacteria. Genetic transformation is
used in many sectors of everyday life. From agriculture to medical treatments, this biotechnology is
allowing for new findings through science. Three methods of genetic transformation are projectile
bombardment, electroporation, and heat shock. (Weedman 2014) In 2008, Osamu Shimomura,
Martin Chalfie, and Roger Y. Tsien were co–awarded the Nobel Prize as they first discovered, used,
and altered Green Fluorescent Protein (GFP). GFP was extracted from jellyfish ... Show more
The lids were then placed on the tubes, and both tubes were placed in ice. E. coli bacteria cells that
had been grown on a media plate overnight, were then added to each tube and completely circulated
throughout the solution using a sterile loop. Both tubes were then returned to the ice. The pGLO
plasmid was then added to one tube and the other acted as the control, and had no pGLO plasmid
added to the solution. Both tubes were then placed on ice for ten minutes, then both tubes were
submerged in 42°C water for 50 seconds in order to shock the bacteria and allow the membrane to
become more permeable. The tubes were then immediately removed from the bath and replaced in
ice for another two minutes. Luria Broth (LB) was then added to both tubes and left at room
temperature for ten minutes. The pGLO– solution was added to a LB nutrient agar plate and to a LB
nutrient agar plate that had ampicillin present in it. The pGLO+ solution was added to a LB nutrient
agar plate that had ampicillin added to it and to a LB nutrient agar plate that had ampicillin, as well
as arabinose. All four petri dishes were stored inverted at 37°C for 24 hours. (Weedman

Staphylococcus Aureus ( S. Aureus
Introduction: Staphylococcus aureus (S. aureus) is a spherical bacteria which is known to produce a
cytotoxin called Panton–Valentine leucocidin (PLV) which destroys leukocytes, and kills tissue
(Lina et al., 1999). Five percent of strains of Staphylococcus are known to produce the disease–
causing toxin (Lina et al., 1999), but though the amount of PLV–producing strains is somewhat
small, the strains which produce PLV are apparently resistant to vancomycin, an antibiotic
commonly used to treat staph infections (CDC, 2002). The first recorded case of S. aureus resistance
to vancomycin was a reduction in sensitivity to the antibiotic observed in Japan, and has since
spread to the United States (CDC, 2002). The most common source of infection of these drug–
resistant bacteria are actually in hospitals, wherein the patients are exposed to the bacteria and
subsequently infected (CDC, 2002). Because of this rise of selection for vancomycin–resistant
bacteria, finding an alternative antibiotic treatment for this strain of bacteria is imperative. There has
been some consideration of alternatives to vancomycin treatment of bacteria as sensitivity toward
the drug decreases. Though vancomycin is still the treatment standard for methicillin–resistant
bacteria, other compounds such as linezolid have proven to be more effective. Linezolid is a
currently FDA approved compound that disrupts protein synthesis in the ribosomes of bacteria. The
result of this disruption is

The purpose of this lab is to use genetic engineering to...
The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the
plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.
Background Information:
Genetic transformation is the change caused by genes. This transformation includes the insertion of
a gene into an organism, changing one of the organism's traits. There are many other uses for genetic
transformation including the altering of plant genes coding for frostbite, pests and spoilage
resistance. It can also be used to digest oil spills and even alter in gene therapy to transform sick
cells into healthy ones. This particular experiment will include the transformation of the bacteria
with the GFP ( Green ... Show more content on Helpwriting.net ...
If the bacterial cells do accept the plasmid then the cell will have antibiotic resistance to the
antibodies, and the bacteria will survive.
Pre–Lab Questions:
The two most important sequences on the plasmid that will be used are the ORI bacteria and the
antibiotic resistance. The ORI is the origin of the bacteria and is an important part of the bacteria
because it does the job of replication. The antibiotic resistance is equally important because it is
used to see which bacteria take in the plasmid. In the lab there will be two micro test tubes used.
One tube will be labeled +plasmid and the second tube will be labeled –plasmid. Both test tubes will
contain the E. coli bacteria and 0.25mL of calcium chloride (CaCl2), but only the +plasmid tube will
receive 10pL of pGLO plasmid. There will also be four agar dishes used in this experiment. They
will be labeled as follows: LB/Amp+, LB/Amp–, LB+, and LB–. The LB/Amp+ dish will have luria
broth, ampicillin, and the pGLO plasmid. The LB/Amp– dish will contain luria broth, ampicillin,
and will not contain any plasmid. LB+ also contains luria broth, will not contain ampicillin and will
contain the plasmid. The last dish, LB–, will have the luria broth, and will not have neither
ampicillin nor the plasmid inserted. The LB/Amp– and LB– dishes act as the control group because
they will be used to observe changes that occur without the plasmid. The LB/Amp+ and LB+ will
act as the experimental dishes because the plasmid will

Lb/Ara + Pglo Case Study
1. The plate with the bacteria that is most like the original non–transformed E. coli colonies is the
LB/–pGLO plate. The bacteria could grow because there was no ampicillin on the plate, and it
couldn't have been transformed because no pGLO was added. Since the plasmid wasn't added and
the environment was ampicillin free, the bacteria on the LB/–pGLO plate should be pretty much the
same as the original E. coli because nothing was done to it.
2. Transformed bacteria cells would be found on plates that contain pGLO, so the LB/amp +pGLO
plate and the LB/amp/ara +pGLO plate. The pGLO is a plasmid that expresses the gene for
resistance to ampicillin. On the plates that contain pGLO, cells take up the pGLO plasmid and will
be ampicillin resistant. When cells take in a foreign plasmid, they are transformed.
3. Both the LB/amp plates (LB/amp –pGLO and LB/amp +pGLO) should be compared. Comparing
the results on each plate would show whether or not the bacteria was transformed. Bacteria with
pGLO added (LB/amp +pGLO) should be able to grow in the presence of ampicillin because ...
Show more content on Helpwriting.net ...
Bioremediation is the use of bacteria, fungi, some types of transformed bacteria, and other microbes
in the decomposition of garbage and breaking down of petroleum products. An example of where a
transformed bacteria was used to reduce pollution is seen when scientists broke down naphthalene,
an environmental pollutant found in soils that are artificially created, by using genetically altered
pseudomonas fluorescents. This bacteria is stimulated to uptake the gene for fluorescence so that
when it breaks down the naphthalene it produces light. The light produced depends on the amount of
chemical the bacteria breaks down, allowing scientists to monitor the efficiency of the process. The
Exon–Valdez oil spill is a famous example that involves the use of transformed bacteria that was
genetically engineered to breakdown hydrocarbons in

The Effect Of Tye Ampicillin Glucose Agar Plates
TYE ampicillin glucose agar plates: 15g Agar, 8g NaCl, 10g bacto–tryptone and 5g yeast extract
should be dissolved in 800ml water (deionised). This solution should be autoclaved before cooling
to 50˚C, and adding 1ml of ampicillin solution as well as 200 ml glucose solution before pouring
plates. These can then be stored for up to a month at 4˚C, ensuring to dry in a flow–bench before
use. M9 minimal medium glucose plates: 15g Agar should be dissolved in 800ml water (deionised)
before being autoclaved. This should be cooled to 50˚C, before adding 200ml 5xM9 salts as well as
1ml of 1M MgSO4, 100micrograms of 1M CaCl2 and 1ml of 1mg/ml VitB1. 2xTY medium: 16g
bacto–tryptone should be dissolved, as well as 10g yeast extract and 5g NaCl all within 1L of water
(deionised), before being autoclaved and cooled to ambient temperature. Antibiotic and glucose
solution should be added. Kanamycin solution: 50mg/ml Kanamycin powder should be dissolved in
water (deionised) before being filtered through a 0.2micrometer filter and aliquots of 1ml should be
produced. Ampicillin solution: 100mg/ml Ampicillin should be dissolved in water (deionised) before
filtering through a 0.2micrometer filter, aliquots of 1ml should be produced. NHS–biotin solution:
10mg N–hydroxysuccinimido–biotin (NHS–biotin) in 1ml of dimethylsulfoxide (DMSO). Glycine
solution: Take 100mM glycine at pH 7.4, dissolving 7.5g of glycine in 1 litre of de–ionised water
before adjusting pH to 7.4 and filtering through a

The Importance Of Genetic Transformation
Genetic transformation is when an organism is given genes that it is able to express from the foreign
DNA (Weedman 111). Genetic transformation is used widely today, especially in agriculture where
organisms are genetically altered to meet the food demands from corns color to squash size. When
transferring genes you need a competent cell which is the cell that is going to express the foreign
DNA (Weedman 111). There are three ways to transfer DNA, transformation where the competent
cell takes DNA from the surrounding environment, conjugation where two bacteria join and transfer
DNA, and then there is transduction where virus or vector carries DNA from a foreign organism to
another (hammiverse.com...na). In our experiment we used transduction, using a vector. There are
three ways to make the competent cell permeable. There is projectile bombardment when a gene gun
is used to coat the competent cell with DNA, there is electroporation when electric pulses are used
to increase permeability and then there is heat shock when you rapidly change the temperature of the
environment the competent cell is in from low to high temperatures making the permeability of the
cell membrane more profound (Weedman 111). We used the heat shock method in our experiment to
increase the permeability of E.coli. In the case of our experiment the competent cell is E.coli. E.coli
is known for its ability of horizontal gene transfer which is the transfer of genes from a foreign
organism and is not

Escherichia Coli, Staphylococcus Aureus, And Enterococcus...
Heidi Torres
Ampicillin VS Inhibition Zone Growth of Escherichia coli, Staphylococcus aureus, and
Enterococcus faecalis
Introduction
The variety of bacteria that lives in our world today is endless. With variety of bacteria comes a
numerous amount of adaptations and enhancements that the bacteria develops, including antibiotic
resistance. Escherichia coli, better known as E.coli, is a gram–negative bacteria with several
different types of clones with a variety of effects. E.coli often enters the intestinal tract as soon as an
infant is born and some forms of it are not pathogenic but rather beneficial to the body(Kaper et al,
2004). When infected with pathogenic E.coli, the body often responds, depending of the type of
pathogenic E.coli, with diarrhea, vomiting, upset stomach. etc. The rate at which E.coli is adapting,
is creating a much more difficult job to find antibiotics that can eliminate the growth of this bacteria
(Tadasse et al., 2002). Enterococcus faecalis is an infectious bacteria that often thrives in extreme
environments (McBride et al, 2007). Enterococcus faecalis is an opportunistic gram positive bacteria
that is usually found in the vaginal tract, intestinal tract or the oral cavity. This bacteria has a high
resistance to many "bile salt detergents, heavy metals, ethanol, azide, and desiccation," worries
hospitals due to their variable tolerance (Zhang W et al, 2013). Staphylococcus Aureus is also an
infectious gram–positive bacteria that may cause a

If Genetic Transformation Has The Meaning Of Change Caused...
If Genetic transformation has the meaning of "change caused by genes" and involves the placing of
a gene into an life form in order to modify the organisms characteristic; the progression of placing
genes from one life form to a different is used to assist of a plasmid and the pGLO plasmid codes
the gene used for GFP as well as the gene for resistance to ampicillin. It is used to manage the
expression of the fluorescent protein; hence, the GFP gene is able to be switched on by adding the
sugar arabinose to nutrient medium of the cell, then the bacteria will be able to glow a bright green
underneath UV light when arabinose is within the nutrient agar medium. Hence, then when one
micro test tube +pGLO and –pGLO are labeled and placed into a foam rack and the tubes are open
and using a sterile pipet used to transfer 250 micro liters of transformation solution (CaCl2 ) in each
tube, position the two tubes on ice, pick up 2–4 colonies of bacteria with a loop, submerge the loop
into the +pGLO tube, repeat steps for –pGLO, put in to ice, and put plasmid DNA into the pGLO;
after the pGLO's need a heat shock by placing the cold tubes into the 42 degrees Celsius hot bath for
50 seconds and back into ice for 2 minutes, later insert the 250 micro liters of LB nutrients broth
into the tube and then placing 100 micro liters into the 4 plates, each individual plate contains
+pGLO LB/amp, +pGLO LB/amp/ara, –pGLO LB/amp or –pGLO LB). If bacteria that contains
+pGLO plasmids is resistant to

Bacterial Lawns
1. Did your bacterial lawns contain only one species of bacteria on each plate, based on lawn
appearance? If not, why do you think that is?
I do believe that my bacterial lawns contained only one species of bacteria on each plate, they are
consistent in color and appearance from the bacteria colonies from Experiment 1. There is only one
difference and it involves plate 1, the toilet handle. I was unsure if it was because of the bacteria or
in reaction to the antibiotics but around the Kanamycin ZOI there is a brighter yellow ring around it.
I thought it was strange that only around there was such a reaction but as I said I was not sure if it
was a reaction to the Kanamycin.
2. Which antibiotic was most effective in killing the bacterial lawn on each plate? Which was the
least effective? ... Show more content on Helpwriting.net ...
This means that the bacteria for this lawn have cells that are surrounded by a cell wall which
includes a thin layer of peptidoglycan and surrounding that is an outer lipid membrane that does not
contain the Gram strain. Yet at the same time while Kanamycin was the most effective the
Ampicillin and Penicillin had reactions as well. The Ampicillin would kill both Gram–positive and
Gram–negative so this could indicate that there are Gram–negative bacteria in the lawn, but the fact
that the Penicillin worked as well was what was interesting. This means that this bacterial lawn
contains both gram–positive and gram–negative bacteria. Gram–positive are bacteria with cell walls
that contain a thick layer of peptiodoglycan that retains the Gram strain. For plate 2, trashcan lid,
only the Kanamcyin worked which indicates that the only bacteria in this lawn are Gram–negative,
but if that is true why didn't the Ampicillin work at all? Ampicillin works to kill both positive and
negative grams, but there was no reaction this time. Could it be that it needs more

Ampicillin Resistant E. Coli Lab Report
Recombinant DNA Used to Create Ampicillin Resistant E. coli Cells
Shalee Sommer
Life 102
Lab section 32
December 2, 2015
Introduction: Certain cells when placed in certain environments or treatments the cell can take on
genetic information passed on through phages or plasmids. (Weedman 2015) E. coli, or Escherichia
coli, is a bacteria that resides in the colons of humans. (WebMD 2009) E. coli colonies usually
perish in the presence of ampicillin a type of antibiotic designed to kill bacteria like E. coli. Heat
shock is a process that places a cell in cold temperatures and then quickly moving the cell to a hot
bath. (Weedman 2015) This treatment causes the cell membrane to weaken and become more
permeable and can thereby ... Show more content on Helpwriting.net ...
coli cells can pick up foreign DNA and use that DNA to allow the colony to survive in situations it
normally would not have. But only if the E. coli doesn't kill the foreign DNA instead.
1. Weedman DO. 2015.Life 102 Attributes of Living Systems. 7th ed. Minneapolis: Bluedoor,
LLC.p109–116.
2. WebMD . 2009. Webster's New World Medical Dictionary [Internet]. 3. Wiley; [Cited 2015 Dec
1] Available from: http://www.csu.eblib.com/patron/FullRecord.aspx?p=416239
3. Dower W, Miller J, Ragsdale C. 1988. High efficiency Transformation of E.coli by High Voltage
Electroporation. Oxford Journals [Internet]. [1988 May 31, cited 2015 Dec 1] Available from:
http://nar.oxfordjournals.org/content/16/13/6127.short
4. Smith H. 1969. TRANSFER OF ANTIBIOTIC RESISTANCE FROM ANIMAL AND HUMAN
STRAINS OF ESCHERICHIA COLI TO RESIDENT E. COLI IN THE ALIMENTARY TRACT
OF MAN. Science Direct [Internet]. [Cited 2015 Dec 1]. Available from:

The Transformation Occurrence With E.coli And The...
The Transformation Occurrence of Plasmid in Bacterial Growth
Cherline Riche
________________________________________________________________________
Panther ID: 5471556
Crystal Garcia, Joselyn Pozo, Tory Jarett
Biology I Lab 1010––Lab Section U55
Abstract:
The objective of this experiment was to observe the transformation occurrence with E.coli and the
ampicillin resistance gene.When plasmids like lux or pUC18 are added in E.coli, they are more
likely to survive in certain environments that contain antibiotics. Plasmid can carry genes enabling
bacteria like E.coli to survive in harsh conditions. This experiment displayed how plasmid work
when inserted in E.coli with and without ampicillin. In certain agar plates, ampicillin was added
with either the lux plasmid or the pUC18 plasmid resulting in colony growth. In other agar plates,
no ampicillin was added when either the lux plasmid or the pUC18 plasmid was added resulting in
colony growth. If growth occurred from the bacteria when it was added in the Ampicillin agar
plates, then it was determined that the E.coli was transformed successfully and is expressing the
Ampicillin resistant genes.
Introduction: The small, circular DNA molecules that exist apart from the chromosomes in most
bacterial species are called plasmids. Plasmids are not needed for the survival of the host bacterial,
but some plasmids can carry genes that makes bacteria ampicillin resistant like pUC18 or

The Effect Of Puc18 And Lux Plasmids On Ampicillin...
Michelle Trujillo
5702361
Michaela Salisbury
BSC 1010L U60
Effects of pUC18 and lux Plasmids on Ampicillin Resistance of Escherichia coli
Abstract
This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux
plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance,
however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced
into the bacteria's genome. The E. coli cultures were mixed with a calcium chloride solution and
then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA.
The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto
petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there
is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24
hours. The results were rather odd because every single one of the samples grew. Several errors
could have occurred here, cross contamination or possibly an error in preparation as every single
sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin
resistant.
Introduction In this experiment we were meant to observe the transferring of DNA. There are many
ways in which DNA can be transferred into an organism, for example; transformation, transduction,
and conjugation. In our experiment we used

Use Of Antibiotic Resistance For A Large Number Of Infections
Maddie Ward
Period 6
January 22, 2015
Transformation Lab
Abstract:
Ampicillin is a beta–lactam antibiotic which can be used to treat a large number of infections. For
example, Escherichia coli (E. coli) bacteria is terminated by this specific antibiotic. Ampicillin
interferes with the formation of bacterial cell walls and thus kills newly dividing cells that must
form new cell walls. Plasmids contain genes that create antibiotic resistance to their host cell. The
pGlo plasmid contains an Ampicillin resistance gene. Therefore, bacteria that take up the plasmid
and transform become resistant to Ampicillin. To carry out this experiment, my colleagues and I
took four petri dishes containing the bacterial host cells (E. coli) on each and the Ampicillin on two
of those four. Through a tedious process, we added the pGlo plasmid to one petri dish of just the
host cell and one petri dish of the host cell and the Ampicillin antibiotic. When examining the results
of our experiment, we noticed that both petri dishes containing zero Ampicillin entirely submerged
in a lawn of the Escherichia coli bacteria, the petri dish containing no plasmid but some Ampicillin
displayed zero growth, and the petri dish containing the plasmid and Ampicillin showed individual
colonies of the E. coli bacteria glowing under a UV light. The reason this occurred is because the
petri dishes that showed a lawn of bacteria contained no Ampicillin so there was no antibiotic to kill
the bacteria off. The petri

Blue Bacteria Lab Report
There was a mix up with placement of the Plasmid containing Bacteria (experiment's culture) and
control bacteria. The experiment's culture was place in the jar with no ampicillin. As a result,
bacteria with plasmid grew as well as bacteria without plasmid. However, it is possible to identify
the transformed culture because of the blue marker.
On the other hand, the control cells do not have the plasmids with ampicillin resistance and blue
marker so the population of bacteria cells are fewer in the jars with jars with ampicillin and they do
not have blue bacteria.
DISCUSSION
Plasmid DNA is negative and bacterium's cell wall is negatively charged. Thus, as DNA approaches
the cell wall it is repelled. The calcium chloride neutralizes the charge of the bacterium and DNA
therefore allowing the Bacterium cell wall to be in close proximity to a plasmid DNA. The
bacterium undergoes heat shock which is technique of place a bacterium in extreme temperature
with wide range. In the lab the bacteria was place in an ... Show more content on Helpwriting.net ...
These are properties E.coli normal does not have so control bacteria should look white and die in the
presence of ampicillin. The ampicillin marker was irrelevant in this experiment due an error of not
placing the experiments 's data in ampicillin. In the end, data on how the bacterium with plasmids
would react with ampicillin is unknown.
Nevertheless, the evidence is successful at proving the transformation of bacteria is possible because
some cell took in the lac Z gene containing plasmid and were able to produce beta–galactosidase as
a consequence turned blue. There seem to be an equal proportion of blue colony to cells without
plasmids. Without ampicillin degradation, there is an excess of bacteria without plasmids so the agar
jar does not have appearance satellite colony agar

Dna Coding For Ampicillin Resistance And Green Fluorescent...
Abstract This experiment was performed to assess the efficacy of genetic transformations on
bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic
transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and
placing them on various media to observe gene expression via growth and color under UV light. The
transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked
the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of
arabinose, the transformed E. coli glowed green. These results support the previous scientific
understanding of bacterial competency, vectors, and gene expression and support gene
transformations as an effective method to transfer the desirable DNA of one organism into another
organism's DNA. These results can be applied to real world issues such as medical treatments, food
production, and environmental conservation.
Introduction
Genetic engineering is used in health treatments, agricultural applications, and environmental
solutions. Genetic transformations incorporate foreign genetic material into the DNA of a different
organism via a vector, which carries the genetic material. Plasmid DNA is small, round, and
autonomous, due to its origin of replication. In biotechnology, plasmids carry beneficial genes, such
as antibiotic resistance, and also a reporter protein, in this case, Green Fluorescent Protein

Plasmid Vs Ampicillin
After analyzing the data recorded for both the agar plates containing ampicillin and those that did
not, it can be concluded that the data provides enough evidence to reject the null hypothesis. There
is enough evidence to support the alternative hypothesis stating that there is a correlation between
plasmids coding for an antibiotic resistant gene and bacterial growth in ampicillin. When a bacterial
solution containing either pUC18 or the lux plasmid is transformed in an agar plate containing
ampicillin, only those cells which took in the plasmid are able to survive and replicate, forming
individual colonies. Not all cells are transformed though, the chances of a successful transformation
were extremely low. In order to see which cell transformed the cells were tagged according to their
plasmid, in the plates containing pUC18 the only ... Show more content on Helpwriting.net ...
As expected the higher growth was seen in the E.coli cells growing outside ampicillin environments,
this is because they grow freely with only minor risk, such as contamination. Possible errors in the
experiment can have included the difference in the amount of liquid broth added to the E.coli sample
that contained no plasmid and that which contained a plasmid. The sample containing no plasmid
received half the amount of liquid broth (150µl) as that of the other two samples (300µl). Liquid
broth can have incremented the growth in the plates containing plasmid twice as much as on the rest
of the plates. Another artifact which can have affected the results was the time each solution spent in
both the ice and hot water bath. Uneven sharing may have also taken place within the E.coli used for
each plasmid leading to different amounts of colonial growth in the different agar plates used. More
accurate results would have been possible if there had been more variation in both E.coli and
plasmid

Plasmid Transformation Of E. Coli
Plasmid transformation of E. coli using pVIB
Savannah Jacobs
April 4th, 2016
BIO 335 Spring 2016
Dr. Koester Abstract Since bacteria are haploid, asexually reproducing organisms it is important for
these organisms to be able to accept genetic variability into their genome. A process called
transformation, which involves absorbing small segments of DNA from deceased organisms in the
natural world, does this. Transformation can also be mimicked in the laboratory using plasmid.
Plasmids are small segments of DNA that occur in bacteria that allow us to regulate if
transformation was successful. We attempted transformation of E. coli cells using plasmid called
pVIB, which allows for luminescence and resistance to the antibiotic ampicillin, from Vibrio
fischeri, however, we did not achieve a successful transformation.
Introduction
Some organisms, such as bacteria, have the ability to transform into a new form by picking up small
segments of DNA from other organisms. This process is known as transformation and happens quite
often in nature. Quite often organisms die and release their DNA into aqueous environments. The
DNA is broken down but it is a while before it is fully destroyed allowing for bacteria cells and
other organisms to transform their own DNA using the broken down segments from the deceased
organism (Dodd). Transformation is an extremely important step in increasing genetic variation in
organisms that reproduce asexually, allowing for them to make both

Positive And Negative Controls In Bacterial Transformation Experiments

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