This biotechnology lab analyzes the effect of transferring genetic information through the alternation of bacterial gene in E. coli (Spilios, 2014). This alteration occurs through plasmid DNA transcribing the new genetic components into RNA, which will translate into an amino acid (Sadava et al., 2014). This newly transcribed amino acid is an enzyme that will give the transformed E. coli cells an antibiotic resistance, Beta-lactamase (Greenfield et al., 2009). The plasmid DNA of interest will be altered to become more resilient to the antibiotic ampicillin, since beta-lactamase could decompose the ampicillin. In addition to plasmid DNA, the bacteria contain other important features such as reporter gene. This reporter gene will act as an aid when observing the effect of the alteration, since this particular gene can be distinguished when a plasmid with foreign DNA is transferred from one to another (Spilios, 2014). Moreover, the reporter gene being used in this lab, Green Fluorescent Protein, is to determine gene resistance to ampicillin. GFP would be useful in this experiment, since it would glow when arabinose operon is present. Ampicillin is a derivative of penicillin that inhibits bacterial growth by interfering with the synthesis of bacterial cell walls. Since E. coli is gram negative, and ampicillin kills the gram-negative bacteria by synthesizing with the cell wall, E. coli should perish under no transformation. However, the ampicillin resistance gene is the enzyme Beta-lactamase, which is secreted by transformed cells into the surrounding medium where it destroys ampicillin (Dörr, 2010). In order to resist ampicillins, E.coli utilizes pGLO plasmid to protect the cell from ampicillin’s invasion. There are four components to...
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...n ampicillin resistance, and able to decompose ampicillin, while untransformed gene would perish because of ampicillin damaging the bacteria’s cell wall. Moreover, presence of arabinose operon would promotes the binding of RNA polymerase and the genes for GFP are transcribed, and this would result in the bacteria glowing under the UV light. The result of this experiment confirmed the hypothesis. As explained in result section, the transformed bacteria were alive, while untransformed bacteria were dead. Moreover, one that contained arabinose operon grew under the UV light, while one without arabinose operon did not grow under the UV light, since the GFP was not transformed by the arabinose operon. The sample with -pGLO LB was expected to have lawn, since nothing inhibit the cell from growing. On the other hand, the sample with -pGLO LB/amp had no colonies, since the
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
In the case of temperatures the cultures were incubated at each determined temperature. For the UV radiation, cells were exposed to UV light for 10 seconds and then grown in 30oC. For the EtBr treatment, 50ul of EtBr was added to the growth medium and cells were incubated at 30oC. In the case of sunlight exposure, cells were exposed to sunlight directly and grown at room temperature
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. In this experiment, the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO or +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin.
Bacterial resistance to antibiotics has presented many problems in our society, including an increased chance of fatality due to infections that could have otherwise been treated with success. Antibiotics are used to treat bacterial infections, but overexposure to these drugs give the bacteria more opportunities to mutate, forming resistant strains. Through natural selection, those few mutated bacteria are able to survive treatments of antibiotics and then pass on their genes to other bacterial cells through lateral gene transfer (Zhaxybayeva, 2011). Once resistance builds in one patient, it is possible for the strain to be transmitted to others through improper hygiene and failure to isolate patients in hospitals.
Background Information and Research: Inserting a gene from the Aequorea victoria jelly fish into the DNA of rabbits, pigs, and mice genetically modifies them to glow-in-the-dark. The production of specific genes are coded by genes. This particular type of jelly fish naturally glows in the dark because a gene coded for a green fluorescent protein (GFP). The goal of genetically modifying organisms is to have the modified organism produce a protein that has been coded by the inserted gene thus causing the modified organism to express the new trait. Genetically modifying organisms is important because it has had health benefits in the development of vaccines. E.coli is a rod-shaped bacteria that is a part of the Escherichia genus and is commonly found in the intestines. When demonstrating how to genetically modify an organism, E.coli bacteria is commonly used because it is a simple organism whose process for protein production, gene expression, is the same as a complex organisms’ process. In this experiment, a GFP was inserted into E.coli as well as a gene that causes E.coli’s resistance to ampicillin. Half of the agar plates that the bacteria was growing on had ampicillin. Ampicillin kills E.coli, so the successfully modified bacteria will have been grown on those ampicillin plates. Plasmids contain genes that are resistant to antibiotic ampicillin; scientists have used plasmids in the manipulation of genes. Plasmids were used because it is resistant to the ampicillin used, so if the bacteria was
Genetic transformation is a process that modifies bacteria, by introducing new genetic material. In our lab we introduced the pre-engineered pGLO into the E. Coli HB101 K-12 bacteria. This pGLO plasmid consists of the gene for the green fluorescent protein (GFP), the ampicillin resistance gene that inactivates the ampicillin in the LB media and the araC gene that indirectly controls the arabinose digestion enzymes [Fig 1].
In this experiment, E. coli will undergo the transformation process to insert a plasmid (pGLO) coded with ampicillin resistance, the ability to process arabinose, and the ability to synthesis green fluorescent protein (GFP). Ampicillin is an antibiotic that acts as an inhibitor on the E. coli’s ability
On the other hand, cells that have resistance from the start or acquire it later may survive. At the same time, when antibiotics attack disease-causing bacteria, they also attack benign bacteria. This process eliminates drug-susceptible bacteria and favors bacteria that are resistant. Two things happen, populations of non-resistant and harmless bacteria are diminished, and because of the reduction of competition from these harmless and/or susceptible bacteria, resistant forms of disease-causing bacteria proliferate. As the resistant forms of the bacteria proliferate, there is more opportunity for genetic or chromosomal mutation (spontaneous DNA mutation (1)) or transformation, that comes about either through a form of microbial sex (1) or through the transference of plasmids, small circles of DNA (1), which allow bacteria to interchange genes with ease.
Every year, antibiotic-resistant bacteria are threatening more and more people. As much of a problem as it is, many people are not educated on the term drug resistance. Since it is such a growing concern, it becomes confusing as to why drug resistance is occurring and what can be done to prevent it. Because drug resistance is such a health problem, determining what it is, how these bacteria can acquire the antimicrobial agents, and the possible solutions to the resistance are the types of actions that need to be taken in order to have a better understanding of how truly powerful these drug resistant bacteria are.
The world has changed a lot due to globalization. Now the well-being and prosperity of countries and nations is highly dependent on healthy international trade and investment relationships. The key factor underlying this prosperity is their openness to the global economy. While the global market is becoming increasingly integrated, it is important for the European Union and Canada to succeed in all areas of global commerce.
The objective of our project was to determine if different wavelengths of light would affect the growth of the Rhodococcus genus and whether the two bacteria would handle DNA damage from UV light exposure differently. Our hypothesis is that various wavelengths of light would inhibit the growth of bacterial colonies of both Rhodococcus species differently. Also, we believe that Rhodococcus fascians (yellow) is more resistant to DNA damage from UV light than Rhodococcus corynebacterioides (red). To perform this experiment, we first made the bacterial growth medium was made. Then, the process of the dilution of the bacteria is performed to allow the bacteria to be seen easily. The diluted bacteria is spread upon the medium in the petri dishes
Technology shapes the environment and even food foundations. The technology called genetic manufacturing has shaped the nutrition frugality since the first bacterium to be hereditarily reformed in 1973. There are three classifications used within genetic engineering: the plasmid technique, the vector technique, and the biolistic technique. The plasmid method, frequently the utmost used process includes bacteria providing plasmids, a minuscule sphere of DNA (The Jackson Laboratory). The rings that the plasmids emit are duplicating molecular generators within the cell. Plasmids are essentially indispensable to genetically contrived cells in the wildlife. Plasmids deliver an operational way in which characteristics that are not typically within a chromosome can be conceded from one cell to an alternative cell. Very few plasmids acquire genes that encode for enzymes such as penicillin or ampicillin and these materials dissolve antibiotics permitting a vast subsidy to the cell because they now become invulnerable to numerous classes of antibiotics. When these cells enclosing plasmids ceases from living adjacent cells clutch the plasmids and acclimate to the qualities that were attained in the previous transaction. He...