Plasmid Lab

In this lab we learned about plasmolysis. We completed this lab to study the effects of salt and distilled water on elodea cells. We first took a leaf of an elodea plant and placed it on a slide with a drop of distilled water. We then placed a cover slip over the leaf and observed the leaf under a compound microscope with lenses that magnified the cells 100 times and then under a lense that magnified the cells 400 times. We then recorded what we saw by drawing a couple cells in each power. Next, we placed a drop of saltwater on the same leaf and observed it again under the same two magnifications. We recorded what we saw by drawing a the group of cells that we saw. Finally, we added a drop of distilled water to wash out the salt water and repeated the procedure of observing and recording at the two different powers. I knew that salt dissolves in water and that the salt may affect the water levels within the cell. I also knew that when salt was exposed to a plant, the plant wilted. However, I did not have a good understanding of plasmolysis, so I did not have an idea on what was going to happen to the cells during the lab.

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

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.

In this lab, we demonstrated that a plasmid could be inserted into the DNA of bacteria in order to alter the physical characteristics of the bacteria. We used biotechnology to manipulate the genetic code of the bacteria so that it would glow when exposed to a UV light. After a couple of days when the glow of the bacteria started to fade, it became apparent that the operon system that enabled the pGLO gene to take affect was stopping.

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.

Coli bacteria, and mix it with the plasmid. We can select the bacteria that are resistant by introducing ampicillin. Bacteria that are not resistant to ampicillin dies. The screening of the bacteria with the resistance occurs when the bla gene turns on to produce beta lactamase which will kill the antibiotic ampicillin. After this, we can get the plasmid into the bacterial cell through the process of horizontal gene transfer through transformation. This can be done by a cold treatment in an ionic solution, then a heat shock where it increases membrane fluidity to take up the plasmid into the bacteria cell. In order to know if the EPAS1-TD gene product was produced by the bacteria, we have to perform a Western Blot. We do this by extracting the protein and run it through gel electrophoresis to separate the proteins by their size. We then label the proteins with the colored probe based on their size. We can detect the EPAS1-TD gene being produced based on the colored

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].

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

Transformation of Escherichia coli to Form a Recombinant Plasmid Containing Genes for both Ampicillin and Kanamycin Resistance

The purpose of the DNA restriction and electrophoresis lab was to first become familiar with the properties of restriction enzymes and discover that they, along with agarose gel electrophoresis, are used to characterize DNA molecules. Restriction enzymes are used to make cuts or join together DNA fragments. The cuts result in either staggered cleavage, or blunt cleavage. Staggered cleavage results when the breaks are offset. This cleavage usually results in sticky end being produced because for each DNA end that is produced there are extensions with unpaired nucleotide bases. Blunt cleavage results when the breaks in the phosphodiester bonds are right across from each other.

The type of bio-macromolecule that is an enzyme is a protein. The three factors that affect the tertiary structure of a protein is the primary sequence of amino acids, the different kinds of bonds involved in the structure, and different charges of the amino acids. When a protein is denatured, all the non-covalent bonds break apart and release energy. This loses the shape and function of the protein.

The purpose of this experiment was to study the transfer of genetic information on plasmid F’lac by using Escherichia coli. Plasmid transfer was measured by using two different methods. The first one was by using selection and contraselection with three antibiotics: streptomycin(which was replaced by naladixic acid for the second part of the experiment),ampicillin and kanamycin and the second one by using a colour indicator ( X-gal). As significant results, the percentage of transfer for F’lac was higher than the percentage for transposition. Also, the experiment demonstrated that E.coli can quickly acquire resistance to several different antibiotics through the transfer of the F’lac plasmid. It was concluded that significant

It started out as one idea, and then developed into something more valuable as many different layers began to uncover. More and more was discovered, and so it began to spread worldwide, engrossing scientists with their unique abilities. Although, it’s important to take note of the fact that a large portion of this interest began with the discovery of a plasmid to act as a vector. A vector is, simply put, a vehicle for foreign DNA. Vectors insert this genetic material into its system, and relocate elsewhere in order for the DNA to replicate and spread throughout the new location. This information proved to be crucial, for this ‘vector-like’ ability allowed for scientists to mix and match different DNA strands in order to genetically modify certain organisms. In order for scientists to accomplish this task, they must first be able to insert DNA into the plasmid vectors themselves. To do so, a restriction enzyme is used, which, depending on what restriction enzyme is selected, will cut the DNA at certain points in the base pairing sequence. This cutting process is done to both the foreign material, as well as the plasmid vector being used for transport. Once completed, both parts will have split into two “sticky ends,” which are separated pieces of the base pairings that are ready to be connected to a matching pair of DNA. It’s important scientists use the same restriction enzyme for both, in order for the same base pairings to be cut from each, leaving sticky ends that can correspond to the opposite strand of the other organisms DNA. The base pairings of each are then lined up with one another in order to create a singular strands with the separate pieces taking on the role as one singular genetic strand. This is accomplished through the help of DNA ligase, which performs as a sort of glue that bonds corresponding nitrogen bases. Once the insertion into the plasmid is completed, it is then time to

After preparing the target DNA, scientists then insert the insulin gene into the plasmid ring. Prior to insertion, the plasmid must be cut open. Special enzymes are required to cut DNA; these enzymes are generally referred to as restriction enzymes (Rediscovering Biology, 2013). After the plasmid is prepared, scientists insert the insulin gene into the ring. The ring is then closed. The human insulin gene is now recombined with the bacterial DNA plasmid. Now that the bacterial DNA contains the human insulin gene, it’s inserted into a certain variety of bacteria. It’s important for scientists to verify the success of their gene transfer to ensure that it’s capable of being reproduced. To make certain, scientists will insert the insulin gene along with a gene coding for antibiotic resistance onto a plasmid. The engineered bacteria will then be grown in a lab using a special gel that contains the antibiotic and other nutrients required for growth. Only bacterial cells containing the antibiotic resistance gene that survive are considered successful (Recombinant DNA, 2004). Multiple plasmids are inserted into each bacterial cell, improving the chances of success in terms of insulin production. As the bacteria cell thrives and begins to reproduce, its inner processes activate the gene for human insulin prompting insulin production (Kamionka, 2011). As the cells reproduce, so too does the human insulin gene. Finally, these bacteria-produced human insulin protein molecules are

Molecular evolution comes from a change in chemical composition of DNA, RNA and proteins over time that happen because of DNA mutation and selective pressures. These mutations help to lead to evolution changes in organisms and gives genetic variants as well. (Lab Manual 8,9,10 overview,1). These mutations in specific genes can be compared across organisms to see how evolutionary changes differ in genes over time. Not all mutations will affect have on the organisms. Any mutations in that nonfunctioning region, is a neutral mutation. If it is in a functional region it can be harmful or beneficial. Harmful rarely happen in surviving organisms (Lab Manual 8,9,10 overview,2).The amount of cytochrome a amino-acid- sequence that is