Escherichia Colo Transformation

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.

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.

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

The transformation of E. coli using plasmid DNA was a success. The positive control plate had a near lawn of blue colonies growing on the plate. This indicated that the E. coli cells took up the plasmid and became ampicillin resistant. The blue colonies formed because the cells were able to produce β galactoisdase and in presence of X-gal the colonies turned blue. There were light blue colonies formed near the edge of the plate. This could due to the lower concentration of X-gal near the edge of the plate so those colonies were not really blue. In addition, there were too many colonies to count so we estimated the transformation efficiency of the positive control to be around 2000 units/μg. On the other hand, the negative is shown in figure

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

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.

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

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

In this lab the genetic process known as transformation will be used to make E. coli bacteria cells glow in the dark by adding GFP. Transformation is when a cell’s genotype is changed by adding exogenous DNA. This process results in the cell accepting the added genes and incorporating them into the reproduction process, creating more cells with the new gene. Genetic transformation naturally occurs in some species of bacteria, however in this lab it will be generated artificially (Bacterial Transformation). In order for transformation to take place the bacteria must be competent. This means that the bacterium needs to be ready to take in the added genetic information from its environment. These genes are absorbed through the cell membrane (Genetic Competence in Bacillus Subtilis). However, for transformation to occur, a restriction enzyme is required.

Abstract In this lab of transforming bacteria that was experiment today, I will be identifying the process of bacterial genetic transformation and how to calculate transformation efficiency. The samples that will be used in today’s bacteria will contain samples of E.coli sand inserted DNA plasmid into their genetic sequence. If done correctly the results will show a successful genotypic and phenotypic mutations, which will display fluorescent under ultra-violent lights or show signs to being resistant to ampicillin. This experiment was primarily for the purpose of growing E. Coli bacteria.

The data that my group obtained during this experiment was almost identical to my hypothesis.  On the -DNA LB plate there was bacteria growth because there was no ampicillin to keep the bacteria from reproducing.  On the -DNA LB/amp plate there was no growth because of the ampicillin that kept the bacteria from growing into colonies. The +DNA LB/amp plate had about 234 colonies of bacteria. Not all of the bacteria on the plate were able to grow into colonies but some did because they interacted/picked up the plasmid. Finally, on the +DNA LB/amp/ara transformation plate most, if not all of the bacteria were transformed and, additionally there was also arabinose sugar which activated the araC gene that codes for the green glowing protein which made the bacteria glow up when we shined the UV-light

coli grown on the petri dish containing LB broth, amp and arabinose (ara) that was also exposed to the pGLO plasmid glowed and grew and an average of 121 colonies. This was unsurprising, as the petri dish contained LB broth, with the necessary nutrients for E. coli to grow, this allowed several colonies of E. coli to grow. While amp was added, this strain of bacteria had been exposed to the pGLO plasmid, this means that the ampicillin gene was transferred into the bacterial genome. This allowed E. coli to grow, despite the quantities of ampicillin added to the petri dish. While E. coli was able to grow, it was also able to glow, the phenotype for the gene GFP. The GFP gene is part of an operon. The inducer for this particular operon is arabinose. It turns of the repressor and allows RNA polymerase to transcribe the GFP gene and produce the phenotype of glowing. This is why this particular group of bacteria was able to glow and none of the others

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).

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

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