Observation Of Gene Expression Using Various Plasmids

The instructor split the class into two separate groups one with the plasmid lux and the other with the plasmid pUC18. Group two was assigned to test the lux plasmid. The, Eppendorf, tubes were labeled “C” for the control plasmid DNA and “lux” for the plasmid lux DNA. The two tubes were then placed into the ice bath. Using a sterile micropipette 5 uL of the lux plasmid was added to the tubes labeled “lux” or 5 uL of the control plasmid was added to the tubes labeled “C” for the control plasmid DNA. Eppendorf tubes had 70 uL of the competent cells added to them with a different transfer pipet. All the tubes were then stored in the ice bath for about fifteen minutes. Another test tube was labeled “NP”, which stands for “No Plasmid”, and 35 uL of competent cells was added to each of the test tubes labeled “NP” during the fifteen minutes. Once the fifteen minutes are up, all three tubes were placed into a preheated water bath at 37 °C for about five minutes. To both the lux

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.

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 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

On August 19th, 2015 this experiment was performed, by 6 separate lab groups.The experiment began by measuring 1 Ml of E. coli into a pipette and pump, then placing the bacteria into a culture medium. The E. coli and medium were then swirled together for a period of 15 minutes, until completely mixed. This mixture was then poured into a petri dish and allowed to solidify for 45 minutes. After the 45 minute solidification time, 5 small paper disks were inserted into the dish. 4 of the disk contained treatments of antibiotic and 1 was left untreated. The

While the plate with -pGLO on the LB/AMP agar plate would not have any colonies, and the –pGLO on the LB agar plate would have many colonies of E. coli. The +pGLO on the LB/AMP plate did have two colonies but the only colonies produced on the LB/AMP/ARA plate had a colony not growing on the actual agar plate. The –pGLO on LB/AMP did not have colonies of E. coli growing on it. But the –pGLO on the LB agar plate had many colonies growing on the plate. All of the colonies were clear in color even under the ultraviolet light. Therefore the plate that did not support my hypothesis was the plate with the arabinose sugar, since that was the one thought to grow E. coli do to the ampicillin resistant DNA that was in theory picked up by the E. coli. This plate also should have glowed green under the UV light but instead the colonies remained clear. I hypothesize that E. coli cells recognized the foreign DNA and then destroyed it believing the new DNA would be harmful to the cell. This tendency of bacteria cells to kill foreign DNA could answer be a problem that would explain why we had few colonies that survived in the presence of ampicillin and why none of the colonies in the arabinose glowed green. Also the time the E. coli

E. coli or Escherichia coli is a prokaryotic cell found the in lower digestive track of mammals and other warm blooded animals. E. coli is an easy bacteria to work with as it doubles quickly and is relatively easy to grow, millions of cells can be grown in several hours. E. coli is an ideal bacteria in the lab because it does not require its temperature to be too hot, too cold, or too precise. A general warm temperature is perfect for this bacteria. E. coli is also easy to care for it does not need a specific type of nutrient, in a lab setting it can be feed any agar, making the bacteria over all cheaper to care for. Strains of E. coli can also operate in aerobic or anaerobic environments.

The focus of this lab was to identify which plasmid (PFG or Pglo) was inserted into the E. coli culture. In order for the E. coli to be transformed, the E.coli must first be made competent. The cells were made competent by incubating the cells in calcium (2+) and then in cold temperatures. A quick heat shot opens pores in the cell membrane and allowed plasmid DNA to enter the cells. The plasmid that was inserted into the E. coli culture had antibiotic-reistance gene and a reporter gene. A reporter gene is a gene that is easy to detect, and for this lab it could be Pglo or PFG. The cells were placed in plates that contained antibiotics to kill any bacteria that did not uptake the plasmid, this is necciary because about 1% of bacteria will be transformed. This allowed only cells that up took the DNA to

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

The purpose of this experiment is to show how different concentrations of Ampicillin affect Escherichia Coli Growth and how the bacteria become resistant to the antibiotic drug. Through a series of steps, which involves streaking agar plates with E.coli sample and application of ampicillin to the E.coli sample on the agar plate, the experiment yields a result that supports the hypothesis. The hypothesis acclaims that ampicillin would affect the growth of E.coli; measuring the zone of inhibition approves the claim in the experiment. The measurements of the zone of inhibition indicate that the generation of E.coli expands as the radius reduces. The reduction of the radius shows the E.coli population is reducing and becoming resistant. In other

The purpose of this lab experiment was to measure the population growth of Escherichia coli, or E. coli, a type of bacteria that can be found in the human body, specifically in the large intestine. It works to aid in digestion as well as the absorption of essential nutrients. While E. coli is extremely beneficial to the human body and most populations are safe for humans, some cause disease if they end up in food that is eaten (Trzepacz et al.) Sickness can occur when waste reaches a water supply, eating meat that is not properly cooked, or drinking milk that is not pasteurized properly. When working with this form of bacteria, it is extremely important to keep it contained for safe measure (Belk et al.) Additionally, this bacterium has also been cited as a cause of infections involving the bloodstream that have life threatening consequences. It is increasingly troubling because antibiotics are now becoming resistant (Collington, 2009.) E. coli is like all other bacteria in the way it carries out reproduction, in that it produces asexually through the process of binary fission.

Coli. To be able to activate the GFP gene the presence of the sugar arabinose is required. In order to make the E. Coli grow, the presence of ampicillin, an antibiotic, and pGLO is present, however, if ampicillin is present without pGLO it will stop the growth of the E. Coli because the pGLO would not be present to stop the antibiotic. In the experiment E. Coli was tested with the different proteins and plasmids, testing if the plate would glow or grow. If the E. Coli was tested with ampicillin, pGLO, and arabinose the plate would both glow and grow because of the proteins present, however, if the E. Coli was tested with ampicillin and pGLO the plate would neither glow or grow because the proteins were not

The general problem of this lab is to see how antibiotics affect bacteria. Antibiotic is defined as a medicine (such as penicillin or its derivatives) that inhibits the growth of or destroys microorganisms. There are different kinds of antibiotics; however, not all of them work on every bacteria. In this lab, we are going to test four different substances and measure the zone of inhibition to see how much antibiotic effect each substance has. These substances have code names: P-10, S-10, C-30, and TE-30. The constant variables in this lab were the type and size of Petri dish, amount of bacteria, number of hours bacteria has to grow, and the temperature. The larger the clear area around the Petri dish, the more effective the substance.

coli, and the other was for s. epi. We covered the inside of each petri dish (that had a jelly base) with the bacteria it was designated for. After that, we split both dishes into four sections. In the middle of each section we would put an antibiotic disc. After all four of the antibiotics were placed in each dish with tweezers, they were incubated overnight. The next day, we measured the zone of inhabitance around each antibiotic disc. I had two statements in my hypothesis. If S. epi is gram-positive, then penicillin, streptomycin, and tetracycline will be effective on it because each one reacts with gram-positive bacteria. If E. coli is gram-negative, then streptomycin and tetracycline will be effective on it because each one reacts with gram positive bacteria. I reject the first statement in my hypothesis because only one of the antibiotics was susceptible on s. epi, while the others were resistant. I accept my second hypothesis because both streptomycin and tetracycline were susceptible on the e. coli bacteria. We can relate this experiment to the outside world by testing the effectiveness of antibiotics on certain diseases and infections. This important for the care and treatment of people so they can continue living their lives. If I or a friend every needed to know the effectiveness of a antibiotic against a bacteria I can look up labs similar to this one to make sure we are taking the right

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.