The Effect Of Puc18 And Lux Plasmids On Ampicillin Resistance Of Escherichia Coli

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no ampicillin in its environment and no plasmid treated E. coli served as a positive control for this experiment because it demonstrated how the E. coli would colonize and grow in a normal setting. The cells in the positive control plate grew into lawn colonies because they were not placed into a selective environment or transformed, so they had no need to acquire ampicillin resistance. Two plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the lux plasmid but were placed in an ampicillin free environment. These two colonies grew

Control plasmids lux and pUC18 were introduced into E. Coli through a process of transformation.

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

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

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.

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

The goal of this experiment was to investigate genetic transformation of E.coli through the reaction of organism to the vector pGLO plasmid. As mentioned, the pGLO plasmid contains genes coding for resistance to ampicillin (amp), and genes coding for production of the green fluorescent protein (GFP) which glows under UV light in the presence of arabinose (ara),which serves as a reporter gene. This green fluorescent

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

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector . The vector is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.

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.