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 β-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 37°C for
First, 50 uL of bacterial cells were made competent by being treated with 590 uL of CaCl2 in three different test tubes before transformation of the plasmids took place. In order to enhance the uptake of the plasmid DNA the instructor added the CaCl2 solution to the bacterial cells. The tubes were placed into a cold ice bath for about ten minutes after the CaCl2 solution was added into the test tubes of E. Coli.
Part 2: 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
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.
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
Discussion What was expected that the strains of E. coli that did not have a resistance to ampicillin would not grow. The transformed strain also changed to a blue color when the X-gal was present in plate. The transformed cells also grew because they were free of the ampicillin because they possessed the amp gene that they used as an shield against the ampicillin antibiotic. The transformed cell who turned blue did so because the gene converted the sugar to a blue color but also contained the amp gene to ensure that they grew even when the ampicillin was present. The growth of the colonies on the plates
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.
Escherichia coli, or E. coli, is a common bacterium that can be found in diverse environments all over the planet, including the gastrointestinal tracts of animals and humans. Many of these strains of E. Coli are essential mechanisms in the digestive tract, while others are pathogens that can cause complications
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
Abstract- 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
Discussion Both of the hypotheses mentioned were supported. 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. An additional source of error is by mutation occuring, that could provide a resistence to ampicillin in the –pGLO LB
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
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
Bacterial Transformation (Escherichia coli) Kadian Jones Dr. F. Redway BIO 104- Lab 3 November 11, 2014 Introduction 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
Introduction: Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial