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.
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
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.
Hypothesis: If a GFP gene is inserted into an E.coli cell, then the E.coli will glow in the dark.
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...
Green Fluorescent Protein fluoresces bright green when exposed to UV light. The GFP gene only activates if there is arabinose present. When arabinose is not present, the arabinose digestion genes are inactive and energy will be conserved. However, when arabinose is present the genes activate and start to break down the arabinose until it is all consumed. BAD encodes for the enzymes used to digest arabinose. The araC gene in the DNA map above pairs with arabinose, and up regulates BAD. However, it also negatively feeds back into its own regulation. Bla is the Ampicillin Resistance gene produces Beta lactamase, a protein that confers ampicillin resistance [1].
The purpose of the lab was to transform E.coli using the plasmid pRFP to promote the expression of antibiotic resistance as well as expression of the red fluorescent protein (RFP). The hypothesis was that if the transformation was successful, then the bacteria would express RFP because the arabinose would activate the plasmid’s red fluorescent protein, and show growth because pRFP allows E.coli to grow even in the presence of an antibiotic. The plasmid was combined with a sample of E.coli through the process of transformation. Following transformation, the protein was isolated using hydrophobic interaction chromatography (HIC) and the size was determined using SDS-PAGE. The results showed that the E.coli transformed by the pRFP would thrive and express the RFP in the proper environment.
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
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
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.
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.
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.
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...