Plasmid Advantages And Disadvantages

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 containsDNA 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,
... Get more on HelpWriting.net ...

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 ofDNA 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

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 pGLOв„ў 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 37Вє 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 37Вє 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

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

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 smallerplasmid 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, mostbacteria 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

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

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 Вµl of
distilled water, which acts as the control in this experiment; 2.5 Вµl of undiluted plasmid DNA; 1.0Вµl of undiluted plasmid DNA; and lastly,
1.0Вµl 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 Вµl 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 Вµl of distilled water to the E. coli cells. In test tube B, we added 2.5 Вµl of undiluted plasmid DNA to the E. coli
cells. In test tube C, we added 1.0 Вµl of undiluted plasmid DNA to the E. coli cells. Lastly, in test tube D, we added 1.0 Вµl 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

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 CellsPlate #Plate Type# of colonies
+pAMP/KAN1LB/amp+kan360
+pAMP/KAN2LBLawn
–pAMP/KAN3LB/amp+kan0

–pAMP/KAN4LB

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 theplasmid, 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 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 ... Show more content on Helpwriting.net ...
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

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 ... Show more
content on Helpwriting.net ...
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". 10Ојl
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 В°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

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

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

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 ОІ–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 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

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.

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

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
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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

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

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
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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

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

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

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
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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 37В°C for 24

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.... Show more content on Helpwriting.net ...
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

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/ОјL. 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 GenEluteв„ў Miniprep Kit. The kit is based on alkaline lysis of bacterial cell followed by adsorption of DNA into silica

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

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

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 Helpwriting.net ...

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

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 Ојl of 10–5 and 10–6 dilutions of donor cells were each plated onto MacConkey (MAC) agar plates without streptomycin. 100 Ојl of 10–5 dilution
of donor cells and 10–5 and 10–6 recipient were also plated onto MAC plates with streptomycin. 100 Ојl 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 37ЛљC 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 37ЛљC 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 Ојl of alkaline SDS detergent solution (Birnboim
& Doly, 1979). After

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

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 Helpwriting.net ...
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

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 transformbacteria 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

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 ОІ–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 37В°C for ... Show more content on Helpwriting.net ...
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

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 ... Show more content on Helpwriting.net ...
6. Incubate both microcentrifuge tubes on ice for fifteen minutes.
7. Take both tubes out of ice and immediately place in incubator at 42ЩҐC 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 37ЩҐC

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 smallDNA 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

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
ОІ–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–ОІ–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 ОІ–galactosidase meaning those specific colonies do not contain the plasmid with the CIH–1 insert (non recombinant
E.coli) (Coventry University 2016). Isopropyl–ОІ–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

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

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

Plasmid Advantages And Disadvantages

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