Aequoria + Pglo Lab

This lab investigated the central dogma of molecular biology by studying the expression of jellyfish Green Fluorescent Protein in E. coli (Mardigan, 2011). After transforming E. coli with the GFP gene under the arabinose operon promoter, GFP expression was analyzed with and without arabinose. Induction was seen only in media with arabinose. To further study GFP protein and structure, an SDS-Page or gel electrophoresis was conducted where proteins were charged to allow them to move in an electrical field in terms of their size. To further study the role of the arabinose operon in GFP expression an overnight culture was done where the plate was frozen and incubated to induce optimal protein folding. The GFP was then purified. Centrifugation, use of buffers, and a column was used to separate the GFP and bacterial cells.

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 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

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

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 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.

The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies

This allows for some colonies to pick up the plasmid DNA, allowing them to transform into colonies. The GFP gene is in the plasmid DNA but is not turned on because there is no AraC. The plate labeled ‘LB/Amp/Ara +DNA’ have the plasmid DNA, ampicillin, and AraC. The presence of AraC allows for the GFP to turn on, resulting in the transformed colonies to glow in

How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires

The pGLO plasmid will transform the E. coli bacteria with a gene called GFP that codes for the Green Fluorescent Protein in the genetic code. GFP was discovered in the jellyfish, Aequorea victoria as a green fluorescent light emitted from the jellyfish. It was typically seen in the dark upon its activation and since then has been used in studies relating to genetic transformation. (Chalfie and Tu 1994) The majority of the studies test the many different factors that are required in the transformation of pGLO which will determine the functionality of GFP in the E.coli bacterium. The first experiment in transforming GFP and E. coli was completed in 1994 by Chalfie and was further refined the same year. The experiment proved the importance of using restriction enzymes, and DNA ligase in the process of transforming GFP to identify arabinose as the primary activator, and to identify the ampicillin

The goal of this experiment was to investigate genetic transformation of E.coli through the reaction of organism to the vector pGLO plasmid. As mentioned, the pGLO plasmid contains genes coding for resistance to ampicillin (amp), and genes coding for production of the green fluorescent protein (GFP) which glows under UV light in the presence of arabinose (ara),which serves as a reporter gene. This green fluorescent

In our hypothesis we stated that only the container containing all of the components +pGLO, LB broth, ampicillin, and arabinose would be the one that genetically transformed. In order for the bacteria to grow at a rapid pace all it needed was LB broth but when you added ampicillin, an antibiotic, it killed off all of the bacteria. +pGLO has the gene to resist the antibiotic so when that was added it was allowed to grow but there was no sugar to turn on the glowing protein. Finally, after arabinose, a sugar, was added it turned on the switch located in the +pGLO for the fluorescence and enabled to grow and glow.

The reason why it did not inhabit any change after the experiment was conducted is because it had no plasmids added to support the growth and the ampicillin actually killed the bacteria. The plasmid would have served as the vector to insert the gene into the E. coli. The -pGLO LB plate thrived because there was no ampicillin present. While the +pGLO LB/amp/ara plate ended up glowing under the UV light because the GFP was only able to show with the presence of the arabinose, because it is what activates the operon. The +pglo LB/amp plate contained a few colonies but it did not glow under the UV light due to the absence of the

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 the cells were able to produce β galactoisdase and in presence of X-gal the colonies turned blue. There were light blue colonies formed near the edge of the plate. This could due to the lower concentration of X-gal near the edge of the plate so those colonies were not really blue. In addition, there were too many colonies to count so we estimated the transformation efficiency of the positive control to be around 2000 units/μg. On the other hand, the negative is shown in figure

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.