Escherichia Colo Transformation

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

The purpose of this study was to see whether E. Coli cells would engage in the pGLO plasmid and glow in the presence of four control environmental factors which are arabinose sugar, bacteria, the antibiotic ampicillin, LB nutrient broth and pGLO plasmid DNA. This was tested using four plates, all the plates had E. Coli cells and different environmental factors. The founding was that E. Coli will only fluoresce when bacteria, pGLO plasmid DNA, the antibiotic ampicillin, and LB nutrient broth are present. The result did not support the hypothesis because it stated that, E. coli cells that are exposed to the pGLO plasmid would engage in the plasmid and glow only if the arabinose sugar is present.

Effect of Genetic Transformation of pGLO Plasmid Containing GFP Gene Using Heat Shock on E. Coli Growth and Ampicillin Resistance ¬Introduction Green Fluorescent Protein (GFP) is a gene that codes for the green fluorescence of the bioluminescent jellyfish (Aequorea victoria) and the sea pansy (Renilla reniformus), making the species glow in the dark (Chalfie, 1995). However, the gene for bioluminescence is not expressed without the presence of the sugar arabinose (Weedman, 2015). GFP can be artificially inserted into cells not native to the Aequorea victoria or Renilla reniformus species through genetic transformation using the plasmid, a small circular DNA piece that is used to transfer a gene from organism to organism, pGLO

This is proven by the survival of the bacteria in the LB/amp +pGLO dish, as the same bacteria that lacked the modification were killed by the ampicillin. However, we were unsuccessful in genetically transforming bacteria to be fluorescent. This is because in the LB/amp/arbo +pGLO dish, we were unable to locate any bacteria.

Genetic Transformation of E. coli Using pGLO Plasmid Introduction The bacteria E. coli is a competent bacteria which has the ability to accept foreign pieces of DNA and express them in itself. In this lab will be testing the hypothesis that E. coli is competent and can express foreign DNA by depositing pGLO DNA, which was created from the same DNA that makes jellyfish fluorescent, into the E. coli to make the bacterium glow. We are also testing the hypothesis that the pGLO DNA can make the E. coli resistant to ampicillin.

Coli. The first standard E. Coli has no resistance plasmid while the second strain contains a resistance plasmid with genes protecting it from ampicillin. This standard E. Coli and pAMP (plasmid-Ampicillin) E. Coli were each streaked across plates containing the antibiotic and containing growth supportive Lurithea Broth. The purpose of this lab was to test their growth in each medium. Our hypothesis was that while the ampicillin resistant E. Coli would show growth in both LB and LB-AMP plate, the standard E. Coli would only grow in the LB plate for it contains no resistant plasmids against the

There were two controls in place during this experiment. The first control was the –pGLO LB plate; which showed that without inserting a plasmid into the bacteria, that bacteria would not be able to glow. The second control was the –pGLO LB/AMP plate; which showed that bacteria that did not contain the plasmid containing the ampicillin resistant gene could not survive in an environment with ampicillin preasent. This experiment had two constants; which were, that we incubated all of the bacteria cultures at the same temperature and the fact that we used the same type of bacteria for all of the plates. A potential sources of error in this lab is that the bacteria could have gotten contaminated from being exposed to air and debri falling into the plate.

Our results show no growth whatsoever in those plates containing Ampicillin; this indicates that we encounter an error during our experiment. The agar plate's outcomes and bioluminescent response done by the bacterium that had the plasmid, it can be presumed due to scientific analysis that Escherichia coli is impervious to ampicillin and the plasmid combines itself with the DNA of Escherichia coli according to other experiments and based on science itself. We can predict that the impact of the bioluminescence in the cells of the microorganisms that is infested unmistakably gives affirmation that the plasmid infuses with Escherichia coli's DNA, guarding the cells that changed from dying, viably creating a gainful situation for the bacterial organisms. Since Escherichia coli is a negative prokaryotic call, it is within the phospholipid bilayer and on top of this is a peptide glycan

Through performing bacterial transformation using the pB325 plasmid, the expected result was to see growth on all plates with +DNA (four plates) and two -DNA plates, the plates with no amp added. This was expected since +DNA E. coli were protected from amp and could grow on normal plates. The -DNA plates with amp were expected to have no growth as the E. coli were not protected against amp. The result of this experiment yielded a lawn of bacteria on all LB plates while none of the LB+amp plates yielded any growth. These results suggest that the experiment did not work as intended. This is because transforming the E. coli with pB325 plasmid was expected to offer those cells protection against amp. Thus, it can be hypothesized

Bacteria are the natural world 's unsung heroes. They receive bad rep due to the disease causing individuals like tuberculosis but the vast majority of bacteria on Earth are harmless if not beneficial to both the environment and humans. Take for example E. Coli a well known bacteria that lives within our intestines. This particular bacteria makes vitamins that we need in order to stay healthy. In this experiment we analyzed the changes seen in bacteria when adding to differing DNA plasmids; pUC18 and lux. To arrive to the most accurate results possible we had to have the E. Coli bacteria made permeable to the plasmids. So it was prepared with calcium chloride. One group of bacteria was given the pUC18 plasmid, and the other group was given the lux as well as the pUC18 plasmids. We then proceeded to incubate them in different containers. Some had ampicillin where as some had none. This was done in order to observe the effect of pUC18, and lux growth patterns. A control with neither plasmid was used to keep a basis for our experiments. Bacteria that had transformed and taken in the lux plasmid without ampicilin were able to illuminate, and only bacteria that gained the pUC18 were capable of surviving with ampicillin. The results observed reveal how foreign DNA can be adapted to fit into another cell 's DNA even incorporated to be an optimal part of the cell. This process makes it a very viable manner of being able to mass replicate advantageous genes, that would

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).

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,

By using the ligation mixture formed in experiment 3B, competent cells undergo transformation in this experiment. The cells do not usually exist in a state of transformation ready but they can be made permeable to the plasmid DNA and the capable of transformation are referred as competent. Transformation is the uptake of heritable information in a competent cell. Competent cells care extremely fragile and need to be handle gently to ensure the success of transformation. Water bath is heated to 42oC as heat shock treatment to start transformation. The competent cells containing recombinant vectors can be identified through blue white screening. Cells containing recombinant vectors are able to survive on Luria Bertani (LB) medium supplemented with the antibiotic, penicillin. The cells transformed by recombinant vectors are plating on plate that containing isopropyl β-D-thiogalactopyranoside (IPTG) which as an inducer of the lac promoter, and 5-bromo-4-chloro-3-indolyl- β-D-galactoside or called X-Gal which is a dye that produces a blue colour when