Pglo Transformation Lab Report

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.

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 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.

In this experiment we were meant to observe the transferring of DNA. There are many ways in which DNA can be transferred into an organism, for example; transformation, transduction, and conjugation. In our experiment we used

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.

Transformation is the transfers of virulence from one cell to another, through the transferring of genetic material. It was originally postulated in 1928 through the works of Federick Griffith, a British microbiologist. Griffith observed that the mutant form, non-virulent form, of the bacteria Streptococcus Pnumoniae could be transformed into the normal, virulent form, when injected into mice along with heat killed normal forms. He concluded that somehow the information the dead virulent form had transformed the mutant form into a virulent form.

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

This is proven by the survival of the bacteria in the LB/amp +pGLO dish, as the same bacteria that lacked the modification were killed by the ampicillin. However, we were unsuccessful in genetically transforming bacteria to be fluorescent. This is because in the LB/amp/arbo +pGLO dish, we were unable to locate any bacteria.

One of the most imperative functions in maintaining the development of evolution is the frequency of genetic transformation: the injection of foreign DNA into another organism’s DNA. This term is defined by the actions of a vector, but more specifically by the actions of plasmids and phages. However, in this experiment we are primarily focused on the effect of the pGLO plasmid transformation of GFP on the E. coli bacteria by introducing a second chromosome or a plethora of cloned plasmids. (Bassiri 2011)

For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).

Genetic transformation occurs when genes are inserted into another gene to change the organism’s trait (Weedman2016). In this experiment, we proceeded to transform the E. coli bacteria with a gene that contained green fluorescent protein. The green fluorescent protein is used in experiments because it beams a green color under a UV light (Chalfie2008). Typically, it is used to mark the expression of genes, which is why it serves as the symbol for all gene expressions (Tsien1998). In the experiment, we will be using pGLO as the organisms that will transmit the disease, otherwise known as a vector. The pGLO in the experiment

This experiment was performed to assess the efficacy of genetic transformations on bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and placing them on various media to observe gene expression via growth and color under UV light. The transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of arabinose, the transformed E. coli glowed green. These results support the previous scientific understanding of bacterial competency, vectors, and gene expression and support gene transformations as an effective method to transfer the desirable DNA of one organism into another organism’s DNA. These results can be applied to real world issues such as medical treatments, food production, and environmental conservation.

If a gene that codes for Green Fluorescent Protein transforms bacteria and GFP glows when transformation occurs, then when two micro test tubes have 250 microliters of transformation solution and places in an ice bath, then 2-4 bacteria colonies are added to each tube with a sterile loop; then a plasmid (pGLO) is added to one of the tubes, incubated in ice for 10 minutes, then heat shocked for 50 seconds at 42 degrees Celsius, then back into 9ice for two minutes; then LB nutrient broth is added to both tubes (250 microliters) and set out at room temperature for 10 minutes. Then, 100 microliters of each solution in the tube are added to four

To start the experiment two small plastic tubes were labeled negative pGLO and positive pGLO as the first step in this experiment. A pipet was then used to move 250 microliters of calcium chloride into each tube and the tubes were iced. Bacteria was added using a new sterilized loop each time to the positive and negative pGLO tubes. The loop was twisted around in the tube to ensure that the bacteria was evenly mixed into the solutions and the tubes were iced once again. Another loop was dipped into the tube containing the plasmid and it was removed when there was a visible slight film of residue. The plasmid was dipped into the tube that was labeled positive for the pGLO gene. The two tubes labeled positive pGLO and negative pGLO were iced