The Transformation Of Genes On The Mitochondrial And Phenotype Of The Cell

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

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.

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

Scientists can study and manipulate genes by using precisely engineered plasmids, According to ADDGENE, a nonprofit plasmid repository, plasmids “have become possibly the most ubiquitous tools in the molecular biologist’s toolbox” (“What Is a Plasmid?”). In this experiment, we will use pGLO to genetically transform the bacteria E. coli. pGLO is a genetically engineered plasmid that carries the reporter genes for both green fluorescent proteins (GFP) and the ampicillin resistance. GFP, a protein typically found in the bioluminescent jellyfish Aequorea victoria, exhibits a bright green fluorescence in the presence of blue to UV wavelengths (Mecham); the protein absorbs UV light from the sun and emits it as a lower energy green light, exemplifying the second law of

coli, to express a gene that is normally causes jellyfish to be fluorescent. The plasmid that contains the gene for the green fluorescent protein (GFP) is called pGLO. The type of genetic transformation we used in the experiment was heat shock which made the membrane of E. coli fluid and allowed the pGLO to enter the cell. In order for the E. coli to express the GFP gene there needs to be arabinose presence, which is a sugar that acts as an on switch for the pGLO. Using this information, we predicted the bacteria E. coli will grow and express the GFP on the plate that consists of +pGLO, ampicillin, arabinose, and LB broth (Luria Bertani broth).

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.

Our research on recombinant DNA mainly consisted of two experiments: Transformation and gel electrophoresis. In our first experiment, four microfuge tubes were given to us: pKAN DNA, pAMP DNA, unknown DNA, and a TE buffer without DNA. The two positive controls, pKAN and pAMP, consisted of an antibiotic resistance gene respectively to their name. The pKAN plasmid contained the gene resistance for kanamycin while pAMP carried the gene resistance for ampicillin. The negative control, TE, only contained buffer without DNA. The fourth tube was our unknown plasmid, which was either pKAN or pAMP; and by way of artificial transformation, we would be able to initiate the identification of our unknown plasmid.

Genetic transformation occurs when an organism’s genetic makeup is altered due to the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab the pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the GFP and beta lactamase to the bacteria’s genome which as a result will be modified. To test the effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at least had some growth.

Introduction: We are obligated to construct a pGLO plasmid with a gene from a jellyfish that is encoded with Green Fluorescent Protein (GFP). The Green Fluorescent Protein is an antibiotic resistance gene. The bacteria throughout our experiment of the transformation of pGLO is transformed by the existence of ampicillin; which the primary inducer is to express GFP. Using the LB platelets and the LB-Amp-ARA platelets the plasmid

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The objective of this experiment was to genetically transform E.coli cells to express ampicillin resistance and to produce the green fluorescent protein by using the pGLO plasmid. It was hypothesized that only the cells with pGLO DNA added to the solution would be able to survive in the same environment as the ampicillin, and that only the cells grown in the plate with arabinose would fluoresce bright green. The results supported the hypothesis, showing that the +pGLO LB/amp/ara plate was the only plate that had fluorescent cells and grew. Additionally, that +pGLO cells were the only ones that grew on an LB/amp plate.

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

Depending on the bacteria species, the manner in which transformation occurs varies. Haemophilus influenza is an example that utilises membrane-bound vesicles to capture double-stranded DNA 3. On the other hand, S. pneumoniae can uptake single-stranded DNA molecules after its cells express competency factors 3. Alternatively, scientists in laboratories transform cells, including ones that are not naturally competent, by inducing them to uptake DNA 3. These transformed cells are now known as competent cells for they have been rendered able to uptake DNA from the environment 18. This artificial transformation process is achieved by using a procedure called the heat shock method 18. This transforming method includes adding DNA to cells in the

By using the ligation mixture formed in experiment 3B, competent cells undergo transformation in this experiment. The cells do not usually exist in a state of transformation ready but they can be made permeable to the plasmid DNA and the capable of transformation are referred as competent. Transformation is the uptake of heritable information in a competent cell. Competent cells care extremely fragile and need to be handle gently to ensure the success of transformation. Water bath is heated to 42oC as heat shock treatment to start transformation. The competent cells containing recombinant vectors can be identified through blue white screening. Cells containing recombinant vectors are able to survive on Luria Bertani (LB) medium supplemented with the antibiotic, penicillin. The cells transformed by recombinant vectors are plating on plate that containing isopropyl β-D-thiogalactopyranoside (IPTG) which as an inducer of the lac promoter, and 5-bromo-4-chloro-3-indolyl- β-D-galactoside or called X-Gal which is a dye that produces a blue colour when

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial