Dna Report

In this experiment, we determined the phenotypic capability of an unknown plasmid along with its size. With the use of gel electrophoresis, we analyzed the gel photograph by using a standard DNA marker, Lambda HindIII, and came to a conclusion based on our results.

In this experiment the objective was to transform E. coli with the pGLO plasmid and calculate the transformation efficiency. The hypotheses were that the plate with only LB agar and untransformed E. coli would grow a lawn; the control plate of untransformed bacteria with LB and ampicillin would experience no growth; the transformed plate with just LB and ampicillin would grow colonies of bacteria but it would not glow green under UV light; and the transformed plate with LB, ampicillin and arabinose would grow colonies that would glow green under UV light. The results found supported each of these hypotheses as the bacteria grew as predicted. The

According to Medicine.net (2012), genetic transformation is a process by which the genetic material carried by an individual cell is altered by the incorporation of foreign/exogenous DNA into its genome. Competent cells are able to accept DNA presented by experimental influence or manipulation, and the application of genetic engineering with bacteria can aid in the fight against diseases, allowing individuals to maintain their lifestyles without the threat of certain illnesses like heart disease, cancer or hereditary disorders (McPhersson 2008). Plasmid DNA are small circular double-stranded helixes, and present in the plasmid are ampr (selectable marker gene) and GFP (Green Florescent Protein), (BIO-RAD 2010).

DNA results were that of pB325 plasmid extracted from DH5α E. coli cells. Expected A260/A280 ratio was 1.8 while expected yield was ~1-3ug.

Genetic engineering is the deliberate modification of the characteristics of an organism by manipulating its deoxyribonucleic acid (DNA). Bacteria possess plasmids which can be used as vectors for bacterial transformation. The plasmids can be altered to include donor DNA thus altering the physiological or physical characteristics of the bacterium. Escherichia coli have been found to be the most effective bacterium in genetic transformation and heterologous expression of human genes or proteins. It was hypothesised that Escherichia coli HB101 K12 could be transformed with a pGLO plasmid to express the genes encoded for green fluorescent protein (GFP), the bla gene for ampicillin resistance and the araC gene a regulatory protein in facilitating transcription and therefore the transformation efficiency for this bacteria could be calculated. Bacillus megaterium, E.coli B strain, Erwinia carotovora, Pseudomonas fluorescens, Staphylococcus epidermidis and Streptococcus lactis will also be subjected to transformation with the pGLO plasmid to collect transformation efficiency data for comparison against Escherichia coli HB101 K12. Escherichia coli HB101 K12 were found to have been successfully transformed with a transformation efficiency falling within the transformation protocol of 8.0 x 102 and 7.0 x 103. The incidence of Escherichia coli HB101 K12 on the LB/amp and LB/amp/ara pGLO+ plates indicated that the bacterial colonies present showed resistance to the ampicillin

In the following experiment, Escherichia coli were transformed using six separate agar plates. Transformation occurs when plasmid DNA is uptake into the bacterial cell. The two ways transformation is facilitated is by placing them in calcium chloride (CaCl2) and heat shock. These two methods allowed the bacterial cells to become competent or more open to the uptake of plasmid DNA. The purpose of this experiment was to transform bacterial cells with plasmid containing ampicillin resistance and lux genes. It was hypothesized that the lux genes would have created a light-emitting reaction on the bacteria while the ampicillin genes should have made the bacteria resistant to ampicillin attacks and be able to grow normally. The experiment results

Transformation is a procedure where cells take up foreign DNA from their environment. This can happen in either prokaryotic or eukaryotic cells. A circular self-replicating molecule of DNA known as a plasmid transfers genetic information between bacteria enabling the microbes to gain antibiotic resistance as well as the ability to adapt to a new environment. Bacteria also have the capability to acquire small pieces (at random) of DNA from their environment but this however is said to be inefficient because only a small fraction of cells will uptake DNA and become transformed. Expressions of genes on the plasmid will alter the genotype and phenotype of the cell. Under specific laboratory conditions a cell which is incubated with the plasmid DNA has the ability to absorb the plasmid into its own cytoplasm. Because these natural processes can potentially be modified by scientists, the transformation procedure can be used as a means of treating human diseases to increase the quality of life.

Plasmids were extracted using the three-step alkaline lysis procedure as outlined by Kado and Liu, 1999. The extracted plasmids were then digested using 1 U of each XhoI and XbaI, respectively according to manufacturer’s (New England Biolabs) recommendation and ran on a 1% Tris Acetate EDTA (TAE) agarose gel. 1 μl of BioLine Hyperladder I was used as the DNA marker.

A transformation was performed so a vast quantity of the recombinant plasmid DNA might be obtained. There was a total of six transformations performed from four DNA ligations (excluding the fifth ligation). All six transformations were then incubated on ice for five minutes, followed by a heat shock, which came from putting the transformations in a 42-degree Celsius heat block for two minutes. Afterwards, the transformations were incubated on ice for another two minutes, then placed in a 37-degree Celsius incubator overnight, then plated. All the transformations included E.coli and luria broth, but differed in their other growth mediums. The first transformation consisted of the 1:1 molar ratio of pET-41a(+) to egfp ligation, kanamycin and

Plasmids are small DNA atoms that are found in the nucleus region of a bacterium. They provide an extra assistance to a bacterium that allows them to survive and reproduce in certain environments, such as making the bacteria resistant to specific antibiotics. In the experiment being conducted, plasmids pUC18 and lux will be tested to determine the different types of growth with Escherichia coli present. In order to do this, CaCl2 was transferred into a tube with E.coli and incubated for 10 minutes. Different measurements of micro liters of various substances are added to the test tubes and later incubated for 15 minutes. A separate test tube is collected, labeled and added its corresponding micro liters of the competent cells. Once this is

Plasmid Based Transformation is used to recombine genes that are taken from tow different sources, or species. Cohen and Boyer used genes that provided drug-resistant strains to the E.coli bacteria.. Then they grew the bacterial strains that had the plasmids and later isolated the plasmid DNA. The plasmid DNA were cut by restriction enzymes and paired to the bases of EcoR1. They were later put together by DNA ligase. Then they isolated the

Mark one tube “+” and the other “-”. The “-” tube will include the plasmid. Using a sterile graduated pipet, add 0.25 mL ice cold calcium chloride to each tube. Hold those tubes on ice. Obtain a starter plate of E. coli and using a sterile inoculating loop to transfer one large colony of bacteria form the starter plate to each tube of cold calcium chloride. In order to remove the bacteria from the transfer loop and break them apart, place the loop into the calcium chloride and twirl the loop rapidly. Dispose of the loop in a bio hazardous waste. Then, using a sterile inoculating loop, dip the loop into the DNA stock tube. When you remove the loop from the solution, check to be sure that there is a drop of liquid contained in the loop area. Transfer the liquid (approximately 10 μL to the “+” microfuge tube and twirl the loop to mix the plasmid into the solution. Place both the positive and negative tubes on ice for 15 minutes. While the tubes are on ice, obtain two Luria agar plates and two Luria agar plates with ampicillin. Label one Luria agar plate “+” and the other “-”; do the same for the Luria agar plates with ampicillin. After 15 minutes, remove the tubes from ice and immediately place them in a 42 degrees Celsius water bath for one minute. Remove the tubes from the water bath and immediately place them back on ice for two minutes. Remove them from the ice bath and add 0.25 mL of room temperature tryptic soy broth to each tube with a

In order to make recombinant DNA the insert must be inserted into the plasmid. To do this, more insert than vector should be added to a new tube and to a ligation kit the following should be added: 2µl of warm vortexed buffer 2, 10µl warm vortexed buffer 1 and 1µl of buffer 3. After this the solution should be incubated at room temp for 15 minutes and then the transformation step should be carried out as below.

Several fragments of plasmid DNA are observed in the 1.0% agarose gel. In the undiluted sample, sizes of plasmid DNA are approximately 23kb, 9.7kb, 5.6kb and 2.5kb. In addition, there is a very faint band at size 80kb. In the 10X diluted sample, however, have only 2 bands at approximately 16kb and 6.5kb. The undiluted sample result is most similar to result in lane 2 with 4 bands as well, whereas the diluted sample is similar to the majority.

Each one recognizes a unique sequence of 4-8 nucleotides in a DNA molecule and are cut at specific locations within the site. The cutting is done by the use of restriction endonucleases, also called enzymes. Hundreds of restriction enzymes have been identified. “A bacterium carrying such a plasmid is able to live and multiply in the presence of an antibiotic, while cells lacking the plasmid are unable to divide. Then, because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large quantities.” (A Kuspa, WF Loomis) In the following experiment a recombinant plasmid was created. A restriction enzyme was chosen based on the presence or absence of a gene (LacZ). The LacZ gene codes for an enzyme called beta-galactosidase.