Pglo Bacteria

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.

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

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

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 lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

The purpose of the PGLO lab was to be able to perform a procedure known as a genetic transformation. We used a procedure to transform bacteria with the gene that codes for a Green Fluorescent Protein (GFP). The actual source of the GFP gene that we used in this complicated experiment is the bioluminescent jellyfish Aequorea victoria. This protein causes the jellyfish to glow under a UV light that was provided in the dark. After the transformation procedure, the bacteria showed their newly acquired gene from a jellyfish and produced the fluorescent protein, which as a result, causes it to glow. If the bacteria glowed in the dark, that was the initial sign that the experiment was successful.

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 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

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

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

The instructions tell the cell what to do and what traits to display. In this particular lab, pGLO plasmids was inserted into E.Coli bacterial colonies. This plasmid contained the bla gene, the araC gene and the GFP gene. These genes changed the bacteria by allowing it to be resistant to a particular antibiotic, Ampicillin and also to glow fluorescent green when in the presence of arabinose and when shown under a UV light. The bla gene codes for the beta lactamase enzyme which allows the bacteria to be resistant to Ampicillin, while the GFP gene codes for the protein that glows in the presence of UV light. Also, the araC gene codes for the protein that allows the GFP gene to produce the green glow protein only in the presence of arabinose. When the bacteria was in the presence of ampicillin and arabinose, the arabinose acted as an inducer which led the GFP gene to start producing GFP. This led to the bacteria glowing under UV light. This clearly shows gene expression as the GFP produced a protein after it was induced. I also learned from this lab that a cell’s DNA can be changed in order for it to produce different proteins. I also learned more about inducers, which bind to repressors to allow a gene to produce a protein. Furthermore, this lab also reinforced my knowledge of of how protein synthesis can be stopped or activated. Overall, this lab shows gene expression and is a primary example of how a cell’s traits can be

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)

The purpose of the pGLO lab was to better understand the process of DNA transformation in organisms, and to observe how genes are then expressed after the transformation occurs under specific circumstances. A species of E. coli was used in the experiment as the necessary form of bacteria. The independent variable was the altered plasmids that were placed inside half of the collected bacteria, which contained new genes such as the glow gene (GFP) from Aequorea victoria jellyfish, the “switch” for the GFP, and/or antibiotic resistance (Ampr). The following dependent variable was then what genes the bacteria expressed after it had either undergone transformation or not, which would be if it glowed or survived exposure to antibiotic. Another independent variable was if the antibiotic Ampicillin or arabinose sugar were present in the petri dish the bacteria were later grown in. The dependent variable was if the bacteria survived the exposure to Ampicillin. There were four groups used in each experiment. The positive control group consisted of bacteria that would be grown in an Luria broth (LB) with no additional plasmids or exposure to Ampicillin or arabinose. The negative control group was one consisting of bacteria grown in a LB and Ampicillin dish with no genetic transformation. The two experimental groups were then one group where bacteria underwent genetic transformation and were

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.