Pglo Plasmid Experiment

Pglo Plasmid Experiment
Abstract
The pGLO plasmid is engineered to express green fluorescent protein (GFP) in the presence of the
sugar arabinose as well as the ampicillin resistance gene β–lactamase (bla) (Brown, 2011). Original
E. coli HB101 do not have ampicillin resistance or the GFP gene allowing them to glow under UV
light. In this experiment, we transformed E. coli HB101 with the pGLO plasmid by heat shock to
make the bacterial cells competent, allowing the plasmid to enter the cell (Brown, 2011). The
mixture of bacteria with pGLO plasmid were given recovery time after heat shock, then spread on
LB/amp and LB/amp/ara agar plates. The bacteria mixture with no plasmid added were spread on
LB and LB/amp agar plates and all four plates were incubated at 37°C for ... Show more content on
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E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin
and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of
transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also
expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge
tube, since they are getting the exact same
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A Study On Bacterial Transformation
Bacterial Transformation
Fiona Obas
5155246
U42 Abstract
DNA encodes the genetic instructions for cells to carry out their daily activities. DNA can come in
many forms; plasmids for example are small circular DNA molecules found in most bacterial cells.
Though plasmids may not be essential for the life of bacteria, it can give cells resistance in foreign
environments. For the purpose of this experiment, an ampicillin–resistant plasmid is introduced to E.
coli. This is done through a process of genetic engineering called transformation. Transformation
works through the uptake, incorporation, and expression of a foreign gene to alter the genetic code
of a cell. Three conditions are needed for successful transformation: a host, a vector, and a technique
to identify the transformed cells. E. coli is used in this experiment as the host (E. coli is commonly
used in biotechnology due to its rapid rate of growth and short reproduction time). A vector mediates
the transfer of foreign DNA into the host cell. Plasmids are commonly used vectors that will also be
used in this experiment. The procedure of tagging is used in this experiment to differentiate the
transformed cells from those that were not. The learning objectives of this experiment are to:
observe the process of bacterial transformation in an experiment; and demonstrate a change in
phenotype due to uptake and expression of the genes in a known plasmid.
Introduction
In this

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

Biological Transformation Of Bacteria And Pglo Plasmid Dna
Abstract: Biotechnology and Genetic Engineering are highly involved with the genetic
transformation of bacteria with the help of plasmid DNA. Genetic engineering ultimately alters
genetic information using genetic material from another organism. The objective of the experiment
was to accomplish genetic transformation using E.coli bacteria and pGLO plasmid DNA. PGLO
carries an enzyme named β–lactamase that offers resistance to the ampicillin antibiotic. Therefore,
bacterial cells can grow and reproduce using pGLO. Four transformation plates containing E.coli
were segregated into 2 groups of experimental and control groups. Two experimental plates
comprised pGLO, but the control plates did not. One experimental plate contained ampicillin and
pGLO. The second experimental plate consisted of ampicillin and arabinose along with pGLO.
Contrarily, one control plate simply had ampicillin. The second control plate only stored E.coli cells.
The results accepted the hypothesis since only the experimental plate with pGLO, ampillin and
arabinose emitted green fluorescent light under UV light. This confirms that bacteria emit green
flourescent light under UV light only when paired with pGLO and arabinose. However, other
experiments have proven how modifying and using other plasmid DNA material can alter results
substantially. Therefore, bacteria are certainly sensitive to the DNA material that is transforming
them. It is undoubtedly beneficial to use the most advantageous

The Effect Of Plasmid On Bacterial Gene Expression
The Effect of Plasmid Addition on Bacterial Gene Expression
Introduction
In the lab conducted, the main idea stemmed from the topics of genetic engineering and genetic
transformation. By definition, genetic engineering is the "modification of an organism's genetic
composition by artificial means" (Lerner). This often involves the micro–insertion of genetic
material. Genetic transformation is the "genetic alteration of cells resulting from the direct intake of
genetic material through the cell membrane" (Transformation (genetics)). Both differ from other
means of genetically altering organisms due to engineering being able to occur between highly
differing organisms not of the same species, and transformation occurring at a molecular level ...
The purpose of the pGLO lab was to better understand the process of DNA transformation in
organisms, and to observe how genes are then expressed after the transformation occurs under
specific circumstances. A species of E. coli was used in the experiment as the necessary form of
bacteria. The independent variable was the altered plasmids that were placed inside half of the
collected bacteria, which contained new genes such as the glow gene (GFP) from Aequorea victoria
jellyfish, the "switch" for the GFP, and/or antibiotic resistance (Ampr). The following dependent
variable was then what genes the bacteria expressed after it had either undergone transformation or
not, which would be if it glowed or survived exposure to antibiotic. Another independent variable
was if the antibiotic Ampicillin or arabinose sugar were present in the petri dish the bacteria were
later grown in. The dependent variable was if the bacteria survived the exposure to Ampicillin.
There were four groups used in each experiment. The positive control group consisted of bacteria
that would be grown in an Luria broth (LB) with no additional plasmids or exposure to Ampicillin
or arabinose. The negative control group was one consisting of bacteria grown in a LB and
Ampicillin dish with no genetic transformation. The two experimental groups were then one group
where bacteria underwent genetic transformation and were

Why Is Genetic Engineering Beneficial?
Introduction:
Biotechnology is an area of science in which nature is used for the benefit of humans. In
biotechnology, scientists manipulate nature to better suit it for human application. One way that
scientists manipulate nature is by genetically engineering living organisms. Genetic engineering can
help humans by curing diseases and making food more nutritious among other benefits. Genetic
engineering is accomplished by taking genes from one organism and planting them in another. In
order to genetically engineer, different things are required depending on the organism being
engineered. For example, bacteria is an organism that can be genetically engineered. To genetically
engineer bacteria, scientists need bacteria, a plasmid, a stressor, an energy source, selection, and
regulation. The bacteria is the nature being manipulated. A plasmid is a small DNA molecule that
when inserted into a bacteria (or other cell) can change the cell's DNA by adding to it. A stressor is
an external stimulus of some type which causes a stress response. Energy is necessary for any
cellular work, so it is necessary for the manipulation of bacteria. Selection shows a selectable
marker, which is a favorable trait and also shows whether the DNA has been successfully planted in
the bacteria. Regulation shows how a trait can be expressed. An example of a bacteria that can be
genetically engineered to glow using these factors is E. coli. The plasmid in this case is pGLO,
which gives the E. Coli a

Pcr Lab Report
Introduction
In this experiment, LacZ DNA segment was isolated by PCR and ligated to pET32a vector plasmid
for further use in future experiments. LacZ gene has been used in wide range of experiments in the
Biochemistry field. The gene produces the –galactosidase enzyme that is malleable and easy to
manipulate, making it a great subject for experiments to understand the molecular genetics of
prokaryotes (Serebriiskii and Rose et al.). The LacZ gene was isolated from E.coli bacteria by PCR
because it is an efficient and affordable method of amplifying a specific gene. The PCR product was
ligated to a vector plasmid, pET32a, so it can be inserted to a bacteria cell to be expressed and
analyzed. Restriction digestion with the same enzyme was done to the LacZ gene and vector
plasmid prior to ligation. This step ensures that the two will ligate at a specific position.
Methods ... Show more content on Helpwriting.net ...
The primers chosen have similar melting temperature of 48.5 C and 49.73 C for forward and reverse
primers, respectively (Appendix). In this experiment, 100L of master mix reaction is made prior to
addition of DNA template. The master mix was aliquoted into 4x20L reactions in different snap cap
tubes. In the first two tubes, 2L of DNA from two different bacteria colonies were added to each
reaction. The DNA is prepared by mixing a tip of BL21 e.coli bacteria colony from a plate in 10L of
dH2O. The third tube was assigned as a positive control and 2 L of control DNA template was
added. The last tube was assigned as a negative control with no DNA template

What Is A Transposon?
Transposition Mutagenesis: Post–lab Questions
Rebecca Herbert
Friday Lab Section
1a. What is a transposon?
A transposon is a section of DNA whose location can be moved, or transposed, from a plasmid to a
chromosome, or vice versa. Transposons are necessary if recipient DNA are missing a sequence that
complements the donor DNA. Also referred to as "jumping genes," transposons are unlike typical
DNA which usually does not move around, and are flanked by inverted repeat sequences which
contribute to their ability to move around.
1b. What is the transposon in this experiment?
The transposon in this experiment is contains kanR in between the inverted repeats on either end,
which will be transposed from the plasmid pVJT128 to the chromosome of the recipient bacteria.
2a. In the first part of the experiment, why did we put the donor on a plate containing nalidixic acid?
In the first part of the experiment, we plated donor bacteria, which was chloramphenicol resistant,
on a Nal plate. Because no donor bacteria would grow on a Nal plate, this was a way of ensuring
that the sample of donor E. Coli bacteria was pure.
2b. Why did we put the recipient on a plate containing chloramphenicol?
Similarly, the recipient E. Coli bacteria is resistant to nalidixic acid, and would be able to grow on a
Nal plate, but not on the Cm plate. By plating the recipient bacteria on chloramphenicol, we can
ensure that the sample was purely recipient if there is no growth.
3. How will we recognize a

Green Fluorescent Protein Lab Essay
Title: Purification of Green Fluorescent Protein
Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria.
Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel
electrophoresis is used to check and analyze the pure protein. Research scientists use Green
Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and
multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC.
Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).
Purpose: To illustrate the process of transformation and perform it using Green Fluorescent Protein.
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Invert.
16. Microcentrifuge for 1 minute. Use a micropipette to transfer the supernatant containing the
recombinant GFP to a new 1.5mL Eppendorf tube.
17. Observe the GFP under ultraviolet light.
Data: (Laboratory Procedure for pGREEN)
1./2./3. LB–plasmid: LB+plasmid: LB/Amp+plasmid: LB/Amp–plasmid
Prediction: average growth average growth growth no growth
Reason: control control it has LB/Amp and plasmid no plasmid
Result: lawn lawn 17 colonies no growth
4.
LB+plasmid (Pos Control) lawn LB–plasmid (Pos Control) lawn
LB/Amp+plasmid (Exper) 17 colonies LB/Amp–plasmid (Neg Control) no growth
5.
a. LB+plasmid and LB–plasmid: Both of these plates had a lawn of bacteria. This proves that the
bacteria are capable of growing on the agar and that there was nothing preventing growth beside the
ampicillin.
b. LB/Amp–plasmid and LB–plasmid: The LB/Amp–plasmid had no growth compared to the LB–
plasmid which had a lawn. This proves that transformation cannot take place without the plasmid, or
DNA.
c. LB/Amp+plasmid and LB/Amp–plasmid: The LB/Amp–plasmid had no growth, but the
LB/Amp+plasmid had growth. This shows that the bacteria was transformed and

Genetic Transformation Of Genetic Dna
Genetic transformation occurs when an organism's genetic makeup is altered due to the introduction
of new genetic information which is then incorporated into the organism's genome. In this lab the
pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the
GFP and beta lactamase to the bacteria's genome which as a result will be modified. To test the
effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first
containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and
another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one
with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not
contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate
would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the
plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates
containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at
least had some growth.
Introduction
Transformation is the directed modification of a genome by the external application of DNA from a
cell of different genotype (Griffiths and Miller).Bacterial transformation is the easiest type of
transformation to create and study due to the single cellularity of bacteria and its

The Transformation Of Bacteria ( Green Fluorescent Protein )
Transformation in bacteria is something that could be essential for survival in a bacteria. In order to
perform this transformation naturally a bacterium must considered competent, otherwise it must
undergo an artificial transformation. Being a competent cell means that the bacteria can take up
DNA from its environment naturally (5). Those that are not competent such as Escherichia coli that
are not naturally competent can be tested with an artificial transformation, such as what we will use
in this experiment. Methods used can obtain things such as chemical mutagens or radiation (1). The
gene used for the transfer is the GFP gene (Green Fluorescent Protein), which gives an illuminating
appearance under a UV light when conducted properly, ... Show more content on Helpwriting.net ...
Experiments introduce the idea that the uptake of DNA by competent bacteria can be the result for
survival reasons such as need for food or evolution purposes (5). The difference between natural and
artificial gene transformation in a bacteria, however, with those who are incompetent versus
competent allow he or she to conduct an experiment with artificial transformation. By doing this one
may be able to determine and observe the formation of biofilms or mutations by transferring a gene
to another organism for beneficial or harmful purpose. This could lead to finding resistants or if a
gene introduced has any effect on the bacteria. In the experiment, the pGLO plasmid contained
encodes the gene for GFP as well as a gene for resistance to an antibiotic allowing a transformation
to take place when adding a carbohydrate such as arabinose to the medium. Research has been
found on the horizontal gene transfer on how virulence factors are acquired as well as genes
resistant to antibiotics spread with microorganisms (5). By conducting a similar experiment he or
she can observe similar findings or observe the results and conduct some sort of understanding of
these ideas. The findings in this experiment are to show a positive result in what causes growth and
the glowing when the plasmid pGLO is added to the positive as well as the difference in the
medium. Understanding the way artificial

How The Genetic Transformation And Determine The...
Aim
The aim of the experiment was to observe the genetic transformation and determine the
transformation efficiency of the bacteria, E.Coli.
Introduction
Injection of DNA into cells which results in the inheritance of traits contained in the DNA injected is
known as genetic transformation (Lorenz et al, 1994). Genetic transformation that occurs naturally
has resulted in evolution. The efficiency and rate of exchange of genetic material can be increased if
both organisms use the same system of genetic information storage e.g. DNA plasmid (Johnsborg et
al, 2007). The bacteria E.Coli which are used in the experiment are sensitive to antibiotic, they do
not glow and are easily transformed (Dickson, 2008). Bacteria are single celled which increases the
result of an uptake of substance, and E.Coli have circular DNA or better known as plasmids. These
plasmids can be replicated and passed on to the next generation. The plasmids added to the colonies
will be used as the medium for insertion and expression of the foreign DNA sequence (Cold Spring
Harbor Laboratory, No Date). A gene carrying the GFP or Green Fluorescent Protein is used in the
experiment. GFP is found naturally in the North American jellyfish, Aequorea Victoria, and is
harmless hence leading to its usage as a tool to study gene expression in an organism as they are
easily visible (Dickson, 2008). GFP causes an organism to glow under UV light.
Escherichia Coli or E.Coli is considered as a gram–negative bacteria.

Observation Of Gene Expression Using Various Plasmids
Observation of gene expression using various plasmids (pUC18/lux), and their role in E. coli
transformation
Nawaz Rahman
Panther ID: 5029032
Signature:____________________________________
Lab Partners:
Manuel Vera
Giselle Janoura
Jeniffer Marranca
Section U17
Abstract
Small circular pieces of DNA molecules located inside the nucleoid in bacterial species
(prokaryotes) are known
as Plasmids.Plasmids do not dictate the survival of the host bacteria, but it can give rise to certain
genes being
activated, if required, providing certain benefits. Survival and reproduction in different
environments can be considered
as one of these benefits. In this experiment we add different ... Show more content on
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Coli
forming into its own small colonies. No growth is also a possible outcome.

The experiment contained a total of six different petri dishes, each containing different treatments to
the E. coli.
The first petri dish held ampicillin resistance gene, luria broth and E. Coli. This served as a positive
control since there
was basically nothing to stop the E. Coli from growing. The petri–dish containing E.Coli, Luria
Broth and ampicillin
with no plasmids provided us with the opposite results. There was no growth. Since there were no
plasmids in the dish
all the E. Coli died hence showing no growth.
In general E. Coli growth was directly proportional to the presence of ampicillin. If it so happens
there was
ampicillin in a petri dish with either the lux or ampicillin resistance gene, the result would depict a
colonial growth.
This is because the ampicillin and plasmid attack one another hence some die and the others live. On
the other hand if
there no ampicillin presents the petri dish would depict a lawn growth, because there is no
ampicillin there to replicate.
Introduction
We began the experiment by adding plasmids, a vector, to the bacteria E. Coli to see the E. Coli
growth with the plasmids in its DNA. Our results will conclude for us the correlation between the
transformation of a plasmid and the growth of E. Coli. The process of

Plasmid Advantages And Disadvantages
Plasmids are small, circular, DNA molecules within a cell. They are not connected to the
chromosomal DNA of the cell, which means that they are essentially free–floating in the cytoplasm.
They contain only a few genes and their own 'origin of replication'–a stretch of DNA that ensures
that the plasmid is replicated by its host. A plasmid is an interesting part of the cell as it only
contains DNA that codes for a specific function that is not necessary for the day–to–day survival of
a cell, but only during certain particularly stressful times, but during the remaining times they still
manage to not be discarded by their host cell. Plasmids can be found in unicellular organisms. They
are most commonly located in bacteria, however they can also be observed in algae and yeast. The
plasmid contains a few genes that code for specific functions. For example a plasmid can contain
genes that, when expressed, can let the host utilize a specific nutrient for metabolism of unusual
substances, including the ability to metabolize a toxic substance, or give them resistance to heavy
metals. The plasmid essentially contains genes that can ensure the survival of an organism under
specifically difficult conditions. One of the more interesting traits however, is antibiotic resistance,
which is most ... Show more content on Helpwriting.net ...
It is a couple of genes that code for a toxin and the antidote to that toxin. The toxin is a stable, long–
lived one while the antidote is unstable and disappears fast. This ensures that daughter cells that
haven't inherited the plasmid, but only the toxin and antidote, will die as they have no way to
produce the antidote that has disappeared. Daughter cells that do, however, have the plasmid
continue to survive with a steady supply of antidote. One example is the F plasmid in Escherichia
coli that encodes for the toxin CccdB and the antidote CccdA. (Engelberg–Kulka & Glaser,

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

Homoserine Synthesis Lab Report
The purpose of this experiment was to determine the cloning, transformation, DNA purification and
protein purification of enzyme homoserine transsuccinylase from Streptococcus pneunomiae
(SpHTS). The PCR result showed that there was no band in the DNA gel electrophoresis, which
means the experiment was not successful. The transformation process presented that there were no
colonies growing in the agar plate. The plasmid DNA had been purified and the concentration
measured was 25.2 ng/μL. The restriction digest sample had two visible fragments at 3000 and 3600
base pairs. There were five fractions of protein purification, which indicated five different protein
extraction. The protein gel electrophoresis showed that the flow through fragment ... Show more
content on Helpwriting.net ...
First, PCR technique was used to amplify the SpHTS gene. PCR is a molecular biology technique
for replicating DNA without using a living organism, such as E. coli or yeast (1). The PCR process
was carried in a thermal cycler which denatures the double DNA strands. Then, primers was
annealed to the single DNA strand. Then, 4 primers were used from a given DNA sequence in the
experiment. Primer 1 and 2 were used for PCR sample A with around 500 base pairs, and primer 3
and 4 were used for PCR sample B with around 300 base pairs. Ligation process was followed,
where sticky ends was created by cutting the plasmid and the PCR product with restriction enzymes,
then DNA ligase created a recombinant DNA (1). Then, it was followed by the transformation
process in which the recombinant plasmid DNA was incorporated into E.Coli. The host cells were
replicated and large quantities of plasmid DNA containing ampicillin resistance were generated. The
project was followed by purification of plasmid DNA using GenElute™ Miniprep Kit. The kit is
based on alkaline lysis of bacterial cell followed by adsorption of DNA into silica

Pglo
The focus of this lab was to identify which plasmid (PFG or Pglo) was inserted into the E. coli
culture. In order for the E. coli to be transformed, the E.coli must first be made competent. The cells
were made competent by incubating the cells in calcium (2+) and then in cold temperatures. A quick
heat shot opens pores in the cell membrane and allowed plasmid DNA to enter the cells. The
plasmid that was inserted into the E. coli culture had antibiotic–reistance gene and a reporter gene. A
reporter gene is a gene that is easy to detect, and for this lab it could be Pglo or PFG. The cells were
placed in plates that contained antibiotics to kill any bacteria that did not uptake the plasmid, this is
necciary because about 1% of bacteria will be transformed. This allowed only cells that up took the
DNA to ... Show more content on Helpwriting.net ...
The second method was to purify plasmid DNA from the E. coli culture and physcically analyze the
DNA by digesting the plasmid with specific restriction endonucleases. The restriction endocucleases
used were Bam HI (B), EcoRV (E), and BgIII (G). These restriction endonucleases recognize
specific short DNA squences. These cut plasmids were then placed in a 1% aragrose gel to
determine the band sizes. The expected phenotype for Pglo is that there will be floursenscens light
produced on the LB + amp + Arab plate, where as for PFG the expected phenotype is that there will
be flourcent phenotype only in LB+ Amp + IPIV. For the physical analysis Pglo will produce
fragment sizes in restriction enzyme (E) 5371bp long. Enzyme (B). fragment sized 625bp, 220bp,
and 4,526bp in restriction enzyme (B). Pfg will produce 2 fragments in restriction enzyme (E), one
will be around 3,855bp long, and the other one will be 2,506bp long. In restriction enzyme (B) there
will be only one band about 6361bp long. From restriction enzyme (G) there should be one bad
around 6361bp

Pglo Transformation Lab Report
Tiffany O'Connor November 9th, 2017 Plasmid Transformation
Purpose: Plasmid transformation is the process of transferring foreign exogenous DNA into a host
cell to change its phenotype.
Theory and Background: Plasma transformation can occur naturally when a cell alters its genetics
by taking in DNA from its environment. Genetic transformation can also be induced in a laboratory
setting, called artificial transformation. Artificial transformation is accomplished by choosing a
competent bacterium and introducing a plasmid.
E. Coli is the bacteria used in this lab. The pGLO plasmid is being introduced. This plasmid
contains genes for GFP ... Show more content on Helpwriting.net ...
This is called "Heat Shocking", a rapid increase in heat opens pores on the cell membrane and
propels the DNA into the cell. Immediately following, place the foam rack with the test tubes back
on ice and incubate for two minutes. This incubation time allows the ampicillin resistant gene to
start replicating. After two minutes remove the tubes from the ice. Using a sterile pipet add 250 ul of
LB nutrient to each test tube. The LB nutrient provides the cell necessary nutrients needed for the
cell to reproduce. Incubate the tubes for 10 minutes at room temperature.
Lastly, tap each test tube with your finer to mix the contents. Using a sterile pipet put half of the
suspension from the "–P" on to the LB plate and the other half of the suspension on to the LB/A
plate. Using a sterile cotton swab, spread the suspension evenly around the surface of both plates.
Now put half of the suspension from the "+P" tube onto the LB/AMP plate and the other half of the
"+P" suspension on to the LB/AMP/ARA plate. Using a new sterile cotton swab, spread the
suspension evenly around the surface of both plates and incubate the plates at 37°C for 24

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

Chapter 4 Explain How Bacterial Plasmids Differ From...
Module 1: Assignment 2: Chapter 4
Toi Thomas
Atlanta Metropolitan State College
Fundamentals of Microbiology (BioL 2215) Dr. Vasanta Chivukula
June 06, 2015
1.
2. Explain how bacterial plasmids differ from bacterial chromosomes.
Bacterial plasmids differ from bacterial chromosomes by not being essential to bacterial growth and
metabolism. They are extracellular chromosomes that contain DNA. Bacterial chromosomes are
essential for bacterial survival.
3. Differentiate between pili and fimbriae.
Pili attach two bacterial cells together, while fimbriae attach bacteria to other surfaces.
4. How does polar flagellation differ from peritrichous flagellation?
Polar flagella differs from peritrichous flagella ... Show more content on Helpwriting.net ...
What causes a bacterial cell to run versus tumble?
Chemotaxis (chemical signal) and phototaxis (light stimulus) stimulate the flagellation to rotate
counterclockwise (run) or clockwise (tumble).
6. Compare the cell envelopes of gram–positive and gram–negative bacteria.
a. What function does peptidoglycan serve?
The peptidoglycan provides support to the cells structure.
b. Give a simple description of its structure.
It has a repeating framework of long glycan chains cross linked by amino acids. c. What happens to
the cell that has its peptidoglycan disrupted or removed? The cell will no longer have a
structural/protective support causing it to rupture from internal pressure.
7. List several functions for the outer membrane in gram–negative bacteria. What is the chemical
composition of the outer membrane?
It has a lipid portion: can act as a toxin when released during an infection.
It may function as receptors and blocking immune responses.
It is also composed of poring proteins: can regulate molecules entering or leaving the cell.

8. Discuss a possible mechanism of Gram Staining in terms of differences in structure and chemistry
between the walls of gram–positive and gram–negative

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

Gene Expression And Its Engineering
Introduction Transformation is the process of allowing a cell to be able to accept a gene into itself
and allow the gene to become part of the cell 's whole, thus changing its form and expressing the
gene 's function. This process was first discovered by the British bacteriologist Frederick Griffith,
who was able to transform Streptococcus pneumoniae into a different strain of Strep. in 1928
(1928). This discovery was pivotal in the way we understand gene expression and its engineering.
An example of genetic engineering using transformation that applies to everyday life is the
synthesizing of human insulin, which is a peptide hormone that helps to regulate the amount of
sugar that runs through the bloodstream. In the early 1900s, insulin from other animals was used to
treat diabetes, but human insulin is now able to be easily produced in bulk through genetic
engineering, and has largely replaced animal insulin for the treatment of diabetes. Insulin is made
from cells in the pancreas, and the extraction of insulin from animals used to require taking it
directly from the animal 's pancreas glands. It was not until 1977 that synthetic human insulin was
genetically engineered by Arthur Riggs and Keiichi Itakura, and was achieved by the process of
transformation using Escherichia coli as a host (1977), which allowed for the easy reproduction of
human insulin. Using this method, the production of human insulin became large–scale, faster,
cheaper, and more accessible. A weakened

Plasmid Pglo Lab Report
Lab Report
This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to
replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are
one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the
cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria
into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to
fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which
causes them to glow a green color under a ultraviolet light.
A I started off the Procedure by labeling my Two tubes +pGLO and –pGLO. After that I used my
pipet to put 250 ul of CaCI2 into both tubes. I placed the tubes in some ice and started to select
colonies of bacteria that were "fat". I then ... Show more content on Helpwriting.net ...
I looked at he pGLO through a uv light. Then I put my loop into the pGLO plasmid DNA tube and
made sure that the ring was covered with he solution, then mixed it into the +pGLO. I then place the
tubes back on ice for 10 minutes and I pushed them under the ice. While my tubes were sitting in the
ice I begin Ito label my LB nutrient Agar plates on the bottom. When the 10 minutes is up for the
tubes in the ice, I removed them from the ice and put them into a film holder and placed them into a
heated water bath for 50 seconds.The settings for the water should be 42 degrees Celsius. When the
50 seconds are up, I placed both tubes back on the ice and had them sit in there for 2 minutes. After
the 2 minutes I I removed

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

The Use Of Transformation As Becoming Increasingly Popular...
Introduction
The use of transformation is becoming increasingly popular in use by genetic engineers to bring new
genetic material into a wide range of life forms. In bacterial transformation, DNA (whether plasmid
or chromosomal) is taken from a contributor cell to a beneficiary cell as a section of exposed DNA.
The giver cells should first be lysed to allow the release of the DNA. After discharge from the
contributor cell, huge chromosomal DNA is effectively separated into smaller parts of exposed
DNA. Conversely, the smaller plasmid DNA stays in place as little, roundabout bits of DNA. Cells
equipped for taking up high–atomic weight DNA is said to be competent. To achieve competence
experimentally, most bacteria must be be grown and studied under painstakingly controlled
conditions. Nonetheless, individuals from some bacterial genera, for example, Acinetobacter, are
actually very naturally competent.
An arrangement of genes is expressed when Escherichia coli is exposed to 42C heat, which allows
the bacteria to live at said temperature. The heat shock step is vital for the uptake of DNA and in
order to recover from this, the bacterial cells are incubated for a short time with non–selective
growth media. Nonetheless, because of the low number of bacterial cells, which contain the plasmid
and the potential for the plasmid not to engender itself in all daughter cells, it is important to select
for bacterial cells which contain the plasmid. This is generally performed with

E. Coli Lab Report
Discussion While conducting this experiment many questions came to mind what effect can
plasmids had on the transformation of E. coli and why did CaCl2 had to be used. During the
experiment the solution was used to neutralized negative charges and heat sock was done to
traumatize the cell membrane to get the cell ready for the intake of plasmids. As shown above
observation can be made from the agar plate which describes the affect that plasmid had on the each.
First, with the LB c, LB np and LB lux DNA exhibited lawn growth consequently lux brought about
the bioluminescence effect on plates LB lux. This was the result of the absence of the Ampicillin
antibiotic and the lux together. So, agar plates labeled LB/AMP c and LB/AMP lux, shown colonial
growth which is a clear indication that minimal ... Show more content on Helpwriting.net ...
Colonies appeared 2–3 days when incubated in the dark room and viewing of the plates done at that
time this is because delays may lead to failure in bioluminescence because it decreases with time
once the colonies have formed. Finally, the Agar plate containing the LB/AMP np showed no
growth because of the presence of Ampicillin and no plasmid to make the antibiotic resistant. As
well lux brought about the bioluminescence effect also on plates LB/AMP lux. As human being's
errors are committed every day, so may have been the case. One observation that may have
manipulated the results in the no growth exhibition was the amount of reagent from the pipette and
the measurements were not as accurate as expected. Or reagents may have been lost during the
transfer from the plates. Some stuck on the cell spreader. Another during the HEAT SCHOCK, water
bath had been preheated and may have lost some heat affecting the intake of plasmid. Another factor
upon introducing plasmid pUC18 into the Eppendorf tube and mixed damage can be physical
damage may have been produced since it very susceptible. To correct the errors in future
experiments, the amount of

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

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

Genetic Engineering is a Process of Altering Genetic...
INTRODUCTION:
Genetic Engineering is a process where an organism's genetic material is altered by introducing a
new DNA. It is where a gene is artificially removed from one organism and transmitted into another
using biotechnology. Its goal is to provide with one or more traits that are not found in recipient
organism, giving it the ability to perform a certain action, which the organism in nature cannot.
DNA and proteins are very important aspects in order to understand genetic engineering. DNA is the
genetic material that is found in every organism. It is located in the nucleus of every cell. It is made
up of four complimentary bases, Adenine to Thymine and Guanine to Cytosine. These four bases
help make the genetic code by providing information for the cell and essential proteins that an
organism requires in order to survive. Some proteins act as enzymes that can help speed up the
reaction. The genetic code is similar in every organism which benefits in the process of genetic
engineering. When a gene is transmitted into the "recipient" organism through the means of
transformation, the similarity of the code allows the "recipient' organism to express the specific trait.
Some bacteria use transformation by acquiring the naked DNA from the environment and alter their
genes by attaining new traits.
Escherichia coli, also known as E.coli is a bacterium that lives in the intestine of warm blooded
organisms that causes sickness and several diseases particularly in the weak

Plasmid Transformation Lab
Introduction: Transformation is the process where a foreign plasmid is inserted and to bacteria. The
bacteria eventually intensifies the plasmid and produces a large amount of it. A plasmid is a circular
DNA strand that is utilized for growth and bacteria.
The purpose of this lab was to introduce bacterial growth undergoing transformation and to
understand the process of transformation.
Methods:
Marked One sterile "+ plasmid" marked another "– plasmid"
Use a sterile pipette to transfer 250 microliters of ice cold calcium fluoride to each tube
Place both tubes on ice
Transfer isolated colonies of E coli from the starter plate to the + plasma tube
Put the cells on the loop in the calcium chloride solution and the + plasma tube and spend the loop
and the solution to dislodge the cell Mass
Immediately suspend the cells repeatedly pipetting in and out with the sterile transfer pipet
Return the – plasma tube to ice. Transfer the mass of cells to the – plasmid tube
We're trying to – plasmid to to ice. Both tubes should be on ice now
Use Loop to add one Loop for a plasmid DNA to the + plasma tube. Immerse the full of plasmid
DNA directly into ... Show more content on Helpwriting.net ...
The LB/amp– plasmid petri dish had no growth because ampicillin was incorporated. Ampicillin is
an antibiotic that kills potential growth for a bacteria. The LB–Plasmid petri dish eventually grew a
lawn of bacteria, this dish only had Laurel broth which is food for bacteria, explaining the
substantial growth. We observed circular colonies that only grew on a little corner of the dish. This
awkward growth is probably due to the inaccurate spreading of the cells.The LB/amp+plasmid petri
dish had growth as well because the ampicillin resistance gene was able to grow since the plasmid
contained a gene allowing for antibiotic resistance. The LB+plasmid petri dish had growth because
there was no antibiotic to inhibit

Bacteria Transformation in Biotechnology Essay
Abstract Some bacteria are able to go through transformation making new combinations of genes.
Transformation is a way of gene variability in bacteria. This experiment is based on the
transformation mechanism of bacteria and gene regulation. The bacteria used for the experiment was
Escherichia coli and the genes introduces for the transformation were: gfp and bla by a pGLO™
plasmid. After the insertion of the target genes and growing the bacteria on specialized LB media, it
could be seen that the transformants were positive for the gene expression. The transformed E. coli
on the media appeared fluorescent green under UV light.
Introduction The bacteria used in this experiment is Escherichia coli which is not naturally
competent. E. ... Show more content on Helpwriting.net ...
coli. Right after it the tubes were shook for ten minutes in a 37º C shaker. There were gather 4 petri
plates, one with LB media, two with LB amp(ampicillin), and the last one with LB amp
ara(arabinose sugar). After the 10 minutes each plate was given an aliquot of 100 microliters with
one of the E. coli of the eppie tubes. The LB plate and LB amp had the pGLO– and the other two
plates, LB amp ara and LB amp, had the pGLO+. After this step it's done the plates are prepared to
be incubated at 37º C for two days and reveal the results of the induced transformation.
LAB 9: TRANSFORMATION PROCEDURE
Results The results for this experiment were a bit ambiguous but still recognizable and pretty clear.
All of these plates were seen under UV light. In the LB plate pGLO– , after the incubation, there
was found a lawn of Escherichia coli colonies that looked green because of the light. The LB amp
plate with the pGLO– bacteria, the E. coli did not seem like it grow on it, the media just looked
green. A count of 172 colonies that looked green, was found in the LB amp pGLO+ plate, this plate
had ampicillin. In the LB ara amp media plate there were found 251 colonies of E. coli. In this plate
the colonies looked fluorescent green under the UV light, the only plate. In a scale of growth from
larger to smaller, the first in line would be the LB, then LB ara amp, proceeds LB amp (pGLO+),
and last one LB amp

An Understanding Of Bacterial Transformation By Plasmid Dna
Bacterial Transformation Lab Report
Elizabeth Amanquah
Genetics 311
Monday 2:00–4:50
Jan Trybula
Abstract
This study was undertaken to develop an understanding of bacterial transformation by plasmid
DNA. This study enables one to observe various traits displayed by transformed bacterial cells. Four
experiments were conducted that included (1) Bacterial Transformation, (2) Genomic/Plasmid
extraction, (3) DNA Electrophoresis, and(4) Plasmid /Oxidation EMSA. In this study, a
transformation of the strain Escherichia coli also known as E. coli will be examined with a special
plasmid called pGLO. This study was done to prepare the plasmid into a bacterium and use it to
make large quantities by amplifying it. By allowing RNA ... Show more content on Helpwriting.net
...
The genetic material that is being incorporated into is the bacterial plasmid
In genetic transformation, DNA genes of one organism are added to another organism. The DNA
that has been transmitted to the new recipient of the genetic material is then expressed and continued
(Holmes et al. 1996). By doing this, large quantities of preferable genes can be made. The process of
transformation can be done naturally or artificially. Escherichia coli is a common bacterium this is
single celled allowing the process for plasmid exchange to not be difficult. The bacterium produces
clones allowing the DNA to not be mixed.
A plasmid is a circular deoxyribonucleic acid (DNA) molecule that is able to replicate itself of the
bacterial chromosome and is often the gateway for which a bacteria is able to gain resistant to an
antibiotic. Plasmid contains the promoter of the arabinose operon called PBAD, which is a
functioning unit of genomic DNA containing a collection of genes under the control of a single
promoter. The genes are copied into RNA together into an mRNA strand to encode a single gene
product.
The plasmid DNA will be used integrated into the genome of the E. coli bacteria. To generate the
effects of the plasmids and transformation, the

Plasmid Lab Report
Introduction In this investigation pUC19 plasmids were used as the vector due to its small size of
2686bp, high uptake efficiency by the host and fast replication time. Important features of this
plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication
allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene
and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends
for the CIH–1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin
resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates
that also has the addition of ampiccilin. The lacZ gene encodes the β–galactosidase enzyme which
aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University
2016). The addition of 5–bromo–4–chloro–3–indoyl–β–thiogalactoside (X–gal) in the agar mix acts
as an artifical substrate for the enzyme so it produces blue E.coli colonies when hydrolised by the β–
galactosidase meaning those specific colonies do not contain the plasmid with the CIH–1 insert (non
recombinant E.coli) (Coventry University 2016). Isopropyl–β–D–thiogalactoside (IPTG) an artifical
transcription inducer of the lacZ gene is also added into the agar mix, binding to the repressor gene
and inactivating it. Therefore when the CIH–1 gene is incorporated within the plasmid MCS and
inserted into the E.coli

Gene Regulation For Safety Assessment
Gene Regulation for Safety Assessment:
In addition to the mph gene, few gene regulations are implemented as safety assessment for the
product. In the recombination process of making this product, Ecoli plasmid has been used as a
vector to insert mph gene into host bacteria. We use a plasmid vector from E.coli that has conserved
sequence for Plac promoter and gef suicide gene. Plac promoter is positioned upstream of mph gene
to regulate its expression. Later, Plac promoter will be activated by induction with IPTG.
A suicide system in E.coli plasmid vector is used to reduce the potential risk of gene escaping into
the environment. Genetic transfer from GM bacteria to other bacteria via trans–conjugation will
cause unexpected genetic combinations. This method is implemented by using recipient plasmid
with suicide gene gef from E.coli, with expression controlled by Pm promoter. Pm promoter
sequence, which present upstream of the gef suicide gene will control the expression of gef suicide
gene.
The suicide system did not interfere with the performance of the GM bacteria until its physiological
function was activated by specific signal. In developing this product, we use Methyl Benzoate as the
chemical suicide signal. Following the addition of methyl benzoate, the Pm promoter will be
activated, and lead to the activation of gef suicide gene. This activates the suicide system of the cells
and the cell growth will decreases as the cells die.
Plan
Methods:
These procedures

Lab Partners : Sage Gibson And Vanessa Song
Eunice Assibu
Lab Partners: Sage Gibson and Vanessa Song.
TA: Mian Li
Undergrad TA: Haley linnet burdge
Bio 118–A60
November 10, 2015
Genetic Transformation
Introduction
There were two parts of this lab and part 1 was; Transforming E. coli with the pGreen plasmid and
Part 2; PCR and Electrophoresis. For this lab, a genetic transformation procedure was performed to
introduce a plasmid to another cell and when the cell reproduces it will make a new copy of the
plasmid. And genetic transformation is a process whereby genetic materials that are carried by
individual cells are changed adding foreign DNA into its genome. Also, a"Plasmids are pieces of
double–stranded DNA that can be replicated independently of chromosomal DNA, and normally ...
There are three steps in PCR, and they are; (1) Denaturing– DNA molecules are heated and
separated into two single strands. (2) Annealing– A primer is used to start the process of building a
new strand of DNA. (3) Extension– dNTPs are added to the reaction mixture to build a new
complementary strand to the template strand. By using a primer a new strand grows in 5' to 3'
direction and the template is 3' to 5' and they can be amplified by the original template.
Hypothesis: If DNA is amplified after each cycle then increasing the number of cycles will result in
a lot of DNA. From the four plates #1,2 3 and 4 used, the plate containing the ampicillin and
plasmid (#4) will result in growing the bacteria, secondly will be plate #2 which will only contain
ampicillin and no plasmid. For the rest of the plates will not grow well. 750 bp swathe in lane #2.
Methods and Materials:
Obtained 2 microfuge tubes of transformation solution (containing cold 50mM CaCl2) and labelled
one "+pGreen" and the other one "–pGreen". 10μl of pGreen plasmid was pipetted into the
"+pGreen" tube, transferred a small amount of the E. coli prepared by using an inoculation loop and
stirred each tube and put them on ice for about 15 minutes. Next, a process called transformation
introduced the E. coli. The tubes were placed in a 42 °C water bath for about 50 seconds which
allows the bacteria to expand. Immediately, the tubes were placed in ice water bath for 2 minutes
which made the bacteria to

Transformation Of Escherichia Bacteria And Dna
Transformation of Escherichia coli in different concentrations of Plasmid DNA
Introduction
This report discusses an experiment which students have to transform and plate competent
Escherichia coli in different concentrations of plasmid DNA. This experiment uses four
concentrations of plasmid DNA to perform four transformations. These concentrations are namely, 5
µl of distilled water, which acts as the control in this experiment; 2.5 µl of undiluted plasmid DNA;
1.0µl of undiluted plasmid DNA; and lastly, 1.0µl of plasmid DNA diluted 1 in 10. Transformation
is an important aspect in genetic engineering as it allows for a particular DNA to enter into another
cell. The aim of this experiment is to be able to perform a cell transformation ... Show more content
on Helpwriting.net ...
Then, we placed 100 µl of the E. coli cells into four Eppendorf tubes each and labelled them A, B, C
and D. After filling them up, we placed them into an ice box. After this, we needed to perform the
transformation process by mixing the E. coli cells with different concentrations of plasmid DNA. In
test tube A, we added 5 µl of distilled water to the E. coli cells. In test tube B, we added 2.5 µl of
undiluted plasmid DNA to the E. coli cells. In test tube C, we added 1.0 µl of undiluted plasmid
DNA to the E. coli cells. Lastly, in test tube D, we added 1.0 µl of plasmid DNA diluted 1 in 10 to
the E. coli cells. Then, we used a P100 pipette and mixed all the liquids in all the Eppendorf tubes
by pipetting the solution up and down. Then, we left the Eppendorf tubes along with its contents in
the ice box for twenty minutes. After twenty minutes, we labelled the base of the four agar plates to
match with the appropriate plasmid DNA concentration about to be plated. We also wrote our
initials, the date, and the table number on the base of the agar plate. We then gently lifted up the
cover of the agar plate with just enough space to allow us to plate each tube of cells onto four
different agar plates – one agar plate to one plasmid DNA concentration. We used a spreader to
spread the cells evenly over the surface of the agar, being careful not let the spreader touch

Lab Report of the Experiment of Conjugation of E. Coli
Abstract:Conjugation is a natural occurring process that involves the transfer of DNA from one cell
into another through a physical connection between the cells. In the following experiment, two
strains of Escherichia coli bacterial cells (donor F'lac+strs and recipient F–lac–strr) underwent
conjugation to produce a transconjugant strain (F'lac+strr). MAC plates and streptomycin were
utilized to determine if conjugation had occurred. When plated, the donor colonies appeared red and
the recipient colonies appeared white. The transconjugant plates showed red and white colonies.
Using alkaline lysis miniprep, a DNA plasmid was isolated from the donor and transconjugant
strains and FIGE electrophoresis was used to determine the size of the ... Show more content on
Helpwriting.net ...
Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB
broth.
100 μl of 10–5 and 10–6 dilutions of donor cells were each plated onto MacConkey (MAC) agar
plates without streptomycin. 100 μl of 10–5 dilution of donor cells and 10–5 and 10–6 recipient
were also plated onto MAC plates with streptomycin. 100 μl of 10–4 and 10–5 dilutions of the
conjugation mixture cells were plated onto MAC agar with streptomycin. All seven plates were
inverted and placed in a 37˚C incubator for about 24 hours. The bacterial colonies on each plate
were counted the next day (colony counts seen in Table I).
Donor colonies were picked with a sterile loop and placed into a sterile test tube containing LB
broth. Recipient and transconjugant colonies were also isolated and placed into sterile test tubes
containing LB broth and streptomycin. The tubes were then placed in a 37˚C shaking incubator at
250 rpm overnight.
After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and
centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly
was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution
(Birnboim & Doly, 1979). After

To Develop Botulinum Toxin E ) Insistant Genes?
To develop botulinum toxin E (BoNT–E) resistant genes, the proposed process will require the use
of two plasmid vectors: pEGFP–C2 and pEF–BOS. Both vectors play important parts in introducing
the gene into the PC12 cells, as well as enabling the SNAP–25 to be able to anchor to the intended
membrane. In this process, both plasmids will be introduced separately instead of trying to engineer
a plasmid that performs the tasks of both plasmids. To produce SNAP–25, PC12 cells will be used
as they are mammalian cells from the adrenal medulla of the rat that allow for the study of fast
growing cells that are found in the brain.
The first plasmid, pEGFP–C2 (shown above), contains the gene that codes for more durable SNAP–
25 proteins to be ... Show more content on Helpwriting.net ...
The plasmid is then introduced into the PC12 cells.
The process of producing the desired SNAP–25 begins with the initial feed of PC12 cells. They will
flow into the first reactor where they will be introduced to the SNAP–25 plasmid pEGFP–C2. The
newly transfected PC12 cells will now contain the gene for improved BoNT–E resistance. From this
reactor, there will be a feed into the second plasmid reactor where the cells will be introduced to the
DHHC plasmid as well as kanamycin, which will kill off the PC12 cells that were not successfully
transfected with the pEGFP–C2 plasmid.
From this reactor, a feed runs to a centrifuge where the dead cells will be removed from the feed
while the surviving cells will be sent to a mixer where the PC12 cells will be introduced to
penicillin, which will produce the batch feed of PC12 cells that have incorporated both plasmids.
The feed from the mixer will be centrifuged before sent to the batch feed reactor where the cells will
grow. The assumption that 75% of the cells are transfected each time will be made, meaning the feed
to the first plasmid reactor will contain 3.2 x 1012 cells, the feed to the second plasmid reactor will
contain 2.4 x 1012 cells, and the feed to the batch feed reactor is 1.8 x 1012 cells.
The batch feed reactor also has a feed from the growth medium mixer. The suggested growth
medium for PC12 cells, according to ATCC, is a mixture containing 85% by

Pglo Plasmid Experiment

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Pglo Plasmid Experiment