E. Coli Bacterial Suspension

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

(Biology Dept.). 0.1 ml of E.coli K or 0.1ml of E.coli B was added to the 10 fold dilution. Using soft agar technique, the growth media mixture with E.coli was plated and incubated.

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

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

11. During the 10 minutes, get the LB agar and LB+AMP agar plates ready. Mark your plates with the transformation tube mixture to use (+ or -), the lab group names, and the date on the top of the dishes.

Next heat shocks the tubes for 50 seconds, followed by icing for 10 more minutes. The heat shock increases the permeability of the cell membrane to DNA. Then add 250 milliliters of LB and incubate for 20 minutes. The 20 minute incubation following the addition of LB broth allows the cells to grow and express the ampicillin resistance protein, beta-lactamase, so that the transformed cells survive the subsequent ampicillin selection plates. Plate 100 microliters the + tubes evenly on two plates; 1 of LB and Amp, and one of LB, AMP, and ARA. Plate 100 microliters of the – tubes evenly on two plates; 1 of LB and AMP, and one on LB only.

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

From there the bacterial colonies were transferred into a tube with CaCl2. 3ul of PBY006 were added into a centrifuge tube by using a pipette. The Centrifuge tube was then chilled on ice for 10 minutes. Once the ten minutes were up it was placed in a 42 degree C heat block for 50 seconds. After being chilled and heated 200 ul LB was added into the centrifuge tube.

All of the plates are incubated at 37 ᵒC for 24 hours. In the second part of the experiment, the concentration of the E. coli culture at 37 ᵒC is calculated using Equation

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

In comparison of the cellular morphology of each bacterium in the mixture (table 2) to their respective pure culture (table 3) showed that Bacillus cereus, Escherichia coli, and Staphylococcus aureus are present in the mixed culture as their respective cellular morphology was the same as compared to the pure

The purpose of the two experiments was to determine the fundamental effects that temperature has on the growth and survival of bacteria. During the first experiment five different bacterial broth cultures of Escherichia coli, Pseudomonas fluorescens, Enterococcus faecalis, Bacillus subtilis and Bacillus stearothermophilus were individually incubated at temperatures of 5, 25, 37, 45 and 55°C for one week in an aim to distinguish the effect temperature has on growth and survival of the five different species. After one week they were observed for distinguishable changes by the turbidity showing an indication of bacterial growth, or the clarity an indication of no survival.

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.

A fresh pipette was used to transfer 0.5ml of broth culture of E. coli, to be inoculated, into a tube of molten agar previously boiled to drive off oxygen. The tube was then rolled to distribute the bacteria and allowed time for the agar to harden. The tube was incubated at 37oC for 48 hours. The procedure was repeated for broth cultures of the bacterium Clostridium sporogenes and B. Subtilis.