pBlu lab

The plasmid used is called the pGLO plasmid, which has been genetically engineered. The pGLO plasmid carries the GFP gene. When GFP is produced, the transformed bacterial colonies will glow bright green under UV light. Green fluorescent protein (GFP) is the trait we are primarily looking for in this experiment. For growth, the positive control was the -pGLO LB dish because it had nothing to inhibit its growth, and the negative control was the -pGLO LB/ampicillin dish, because it did not have the genes for Beta-Lactamase to protect it from ampicillin, so it could not grow.

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

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 hypothesis above tested the insertion of the pGlo gene to see if the bacteria, E.Coli, will reproduce and grow in the presence of ampicillin and to see if it will cause a green fluorescent glow. (PGLO™ Bacterial Transformation Kit,2017). Based upon the results from this experiment the hypothesis did support the hypothesis and that the presence of the pGlo gene inserted into the E.Coli did cause for growth and fora fluorescent glow to occur. In the experiment, the petri dishes that contained no pGlo (-pGlo) did not show any reproduction nor did a green glow appeared in both dishes. Unlike the two petri dishes, that contained the pGlo gene and ampicillin, the data data showed both reproduction and a glow in the petri dishes.

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.

In the LB (pGLO negative), it is expected to not see any colonies growing. As a result of this experiment, it shown any growth colonies but this only had shown a large number of white, circular colonies that were found across the surface of the agar. In the LB/Amp (pGLO negative), it is expected to see any growth colonies. In the experiment, the resulted was no growth colonies because this has Ampicillin and no pGLO. Now, the LB/Amp (pGLO positive), it is expected to have growth colonies in the agar plate. As a result, it was shown growth colonies in the agar plate because has positive pGLO regardless having Ampicillin. In the other hand, both positive pGLO have the same components but in one plate was added Arabinose. The LB/Amp/Ara (pGLO

The transformation experiment successfully altered the ampicillin resistance of E. coli through the incorporation of the pGLO

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.

The agar plate labeled ‘LB –DNA’ is the control plate to show what it would look like if the bacteria does not pick up the DNA. Since it does not have plasmid DNA or antibiotics added to it, E. coli grows all over the plate, referred to as a lawn of bacteria. The plate labeled ‘LB/amp –DNA’ has the antibiotic, ampicillin, but still no DNA to resist against the antibiotic. This is why there was no growth of bacteria seen on this plate. The plate labeled ‘LB/amp +DNA’ has the antibiotic and the DNA plasmid, which has the bla gene that codes for ampicillin resistance.

On the plate with +DNA/+Amp, four colonies grew, however they did not glow. The +DNA/+Amp/+IPTG had three glowing colonies grow on the plate. The -DNA plate had E. coli growth but no colonies had formed. The plate with -DNA/+Amp had no E. coli growth. On the second day, the E. coli colonies on the plate with ampicillin and IPTG glowed brighter and there was more growth on the +DNA plate with ampicillin only.

Figure 3 shows the growth of three differently treated E. Coli plates. -pGLO plates are both controls. -pGLO with only LB experienced the most grow because

Of the six petri dishes 2 experienced a lawn, two no growth, and two were isolated. Only one plate experienced fluorescence which was the pGLO + on a medium of LB amp + ara. The pGLO- on a medium of LB experienced a lawn of colonies since an LB medium is perfect for growing E. coli coupled with an incubation of 37 degrees. The presence of ampicillin inhibits cell synthesis in E. coli, with that being said the pGLO- plated with LB amp did not grow due to this (Rogers and Mandelstam, 1962). Both the pGLO + plated with LB/amp and LB/amp/ara were able to grow due to the resistance of ampicillin and the 8 microliters of pGLO added to pGLO + tube. In the pGLO + plated with LB/amp/ara the arabinose caused for transcription of GFP to occur resulting

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.