Expression and Purification of recombinant Green Fluorescent Protein

Expression and Purification of recombinant Green Fluorescent Protein ABSTRACT: The purpose of this experiment was to determine if a His-6 tagged recombinant form of Green Fluorescent Protein could be expressed in a pRSETA vector of E. Coli. This was determined through multiple procedures beginning with purifying the sample with Ni +2 agarose chromatography which showcased the relative fluorescent activity of the samples, which elution sample two (E2) had approximately 100,592.2 RFU/mg . The yield of total protein was found by use of a Bradford Assay and a standard curve. The purity of the GFP was determined by comparing the intensity of bands that appeared at around 31.4 kDa (the molecular weight of rGFP) to a molecular weight ladder on an SDS-PAGE gel. The Western Blot test, utilizing a nitrocellulous membrane, confirmed the expression of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel which E3 had an estimated purity of 0.4, indicating a yield of approximately 29.4 ug of rGFP for the third Elution (E3) after a total p rotein amount of 49 ug was extrapolated from the Bradford assay. INTRODUCTION: The Green Fluorescent Protein was first isolated from the Aequorea Victoria jellyfish and studied by Osamu Shimomura. In other organisms other than this specific jellyfish, there are fluorescent proteins with more color varieties than just green (Ward). GFP is able to fluoresce due to the formation of a chromophore in its center that resides in a stable beta barrel structure. The interactions between Ser64 and Gly67 on an alpha strand form a five member ring structure and its this structure that contains properties for fluorescence. When ultraviolet light is absorbed at 395nm, an emission wavelength of 510nm is seen as fluorescent green light. The excitation wavelength of 395nm excites the electrons within the protein and raises the energy of the protein. As the protein loses energy, it emits the energy at a wavelength of 510nm. Wild type GFP does not have immensely visible fluorescence, therefore a genetically modified form of GFP, GFPuv, was used in order to better observe fluoresc ent activity. For rGFP to be fully expressed, GFPuvs open reading frame was pRSETA vector, which was cut by a restriction enzyme at sites that complimented the start and stop codons of the His-6 tag sequence. A T7 promoter and Xpress Epitope were also genetically added into the vector with the purpose of simplifying purification. T7 polymerase minds to the T7 promoter and activates the production of GFP, while the Xpress Epitope allows for the binding of a primary antibody. The binding of a primary antibody allows for the binding of a secondary antibody which contains horseradish peroxide, producing bands on the nitrocellulose membrane. (2 pg 351) Histidine-6 tags primary purpose is to aid in the purification of rGFP proteins in affinity chromatography. Histidine has an affinity to bind with transition metal ions, and is why a Nickel+2 agarose chromatography was chosen. When a sample is passed through the Nickel+2 agarose column, the His-6 tags fused with rGFP protein will bind to the column and any proteins not of interest or containments will pass through the column. To elute the bound rGFP proteins from the column, elution buffer containing Imidazole is used and competes with the histidine residues for the binding sties to the Nickel+2 agarose column due to its higher affinity for them. Elutions from the column therefore contain rGFP. (2 pg 359) The purpose of this experiment was to see if a His-6 tagged recombinant form of GFP, from the E. coli strain BL21pLysS, could be expressed and then purified using Ni+2 agarose affinity chromatography technology. If this experiment is successful, an inexpensive and relatively simple method to measure gene expression and locate and track proteins may be at hand. MATERIALS AND METHODS: Bacterial Expression Grow two bacterial cultures: G, containing BL21, DE3, pLysS, and the plasmid pRSETA-GFPuv with the GFP sequence, and V, which is identical to G, but it does not have the GFP sequence. Incubate both cultures at about 37Â °C until OD600 equals 0.5. Transfer 1ml of each culture into separate centrifuge tubes and centrifuge to obtain a pellet. The supernatant from the tubes will be discarded and then label the tubes V0 and G0 and stored at -20Â °C. Induce the rest of the culture with IPTG and allow to grow for 3 hours. After the 3 hours, collect 1ml of each culture and centrifuge. Label the tubes V3 and G3 and were store at -20Â °C. Collect 15 ml of G and centrifuge, and label it as G3-15ml and store at -20Â °C. (1 pg 105) Preparation of Crude Extract After the slow freeze process, add 1 ml of breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) to the frozen bacterial pellet G3-15ml, and pipette up and down until completely dissolved. Then transfer the contents to a centrifuge tube and vortex for 5 minutes and place in a 37Â °C water bath for 10 minutes. Afterwards, incubate in dry air at 37Â °C. Centrifuge the mixture at 4Â °C and transfer the supernatant into a clean centrifuge tube and take a small sample to label as GCE. The remaining supernatant will go through the Nickel+2 agarose column. (1 pg 110) Ni+2 NTA Chromatography Pack a 3ml plastic syringe with a small amount of glass wool to prepare the Ni+2 agarose column. After that, pipet 1 ml of breaking buffer into the syringe to remove any air bubbles. As the breaking buffer is going through the syringe, pour some breaking buffer into the leur-lock and then screw it onto the syringe. Add 1ml of 50% Ni+2 agarose into the column and then open the leur-lock to pack the agarose matrix in the colum. Add 5 ml of breaking buffer subsequently into the column to push the ethanol of the Ni+2 agarose through the column. Then close the leur-lock and apply the crude extract to the column and allow 5-10 minutes for the histidine tags on the rGFP to bind with the Ni+2 agarose beads. Open the leur-lock and collect the non-crude extract in a centrifuge tube labeled W1 for the first wash. Add 0.5ml aliquots of breaking buffer to the column and collect the washes in tubes W2-W10. Add 10 increments of 0.5ml of elution buffer and collect in tubes E1-E10. Then store your GC E, W1-10, and E1-10 samples at -20Â °C. (1 pg 111-2) Bradford Assay Analysis To create a Bradford assay, mix protein sample with water, and then add Bradford reagent dye. Vortex the mixture and then transfer it to the wells of a microtiter dish, so that the absorbance of your protein sample can be determined at 595nm using a spectrophotometer. To determine the amount of total protein present in the sample volume, you must create a Bradford standard curve using 0, 2.5, 5, 10, 15, and 20 ug of BSA and the absorbance values observed to determine a best-fit line. Perform the assay in singlicate using samples W1-W6 and E1-E6 to determine if the microplate data falls within the standard curve. Once you have determined what volume of sample to use, repeat the Bradford assays two more times for each sample. Use the standard curve and experimental absorbance values to extrapolate the total protein present in the volume of sample you use. (1 pg 124-6) SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions Create a 12% Resolving Gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x resolving buffer [0.75M Tris pH8.8, 0.4%SDS]. Pour the resolving gel into a gel electrophoresis set up and overlay with some water. Allow the resolving gel to polymerize and proceed to making a 5% Stacking gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x stacking buffer. Pour the stacking gel on top of the resolving gel and immediately insert a comb to form the wells. Prepare your loading samples with 4x sample loading buffer and then vortex, boil, and centrifuge. Do this for the G0, G3, GCE, W2, W3, E2, and E3 samples (or fractions that contained the most rGFP fluorescence). (1pg 111-2) After the gel has solidified, transfer the apparatus into the electrophoresis tank and load the electrophoresis buffer into the tank. Then load your samples into lanes one through seven respectively along with a marking ladder to compare the distances the samples traveled after electrophoresis. Electrophorese at 200V for approximately 45 minutes and then stain your gel with Coomassie Blue dye. To remove the stain that has not been absorbed by proteins, the gel is soaked in dilute acid and methanol. Western Blot Develop an SDS-PAGE gel as previously described using your sample fractions and transfer the proteins onto nitrocellulose transfer membrane using two locking cassette lids with sponges and filter paper encompassing the gel and nitrocellulose. Remove the nitrocellulose and stain the membrane with Ponceau S stain, and allow to incubate for approximately 2 minutes. Once stained, wash the membrane with nanopure water until bands are visible on the membrane. Next, perform the blocking step by placing the membrane in a container containing 5% non-fat dry milk/TBS solution and incubate on a shaking platform. Pour out the blocking solution and add 0.05% Tween 20/TBS solution and then allow it to incubate on a shaking platform, and then pour out the solution. Repeat this wash step two more times. Add mouse IgG anti-Xpress epitope MAb and allow incubate on a shaking platform this is the primary probe step. Then repeat the wash step as mentioned previously three more times, and add Sheep IgG a nti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution (the secondary probe). Allow the membrane to incubate, and then perform the wash step twice. For the final wash, use only TBS, and then add TMB substrate solution and incubate until appropriate banding is shown. Stop the reaction with distilled water and scan or photograph the membrane as the results may fade with time. RESULTS: In the specific strain of E. coli, BL21pLysS, pRSET-GFP represses the lac promoter which is part of the operon needed for T7 polymerase activity. T7 polymerase binds to the T7 promotes of pRSET-GFP and activates the His-6 tag that expresses the fluorescence of GFP. Even though some GFP is produced this way, it is experimentally not enough, so IPTG is added to repress the repressor so an increase of GFP production can occur. V0, V3, G0, and G3 were all bacterial cultures of E. coli with specific distinctions between all of them. V0 and G0 were collected from bacterial cultures that were not yet induced, but G0 contained the GFP sequence while V0 did not. V3 and G3 were the V0 and G0 samples after induction had occurred for 3 hours, and again G0 contained the GFP sequence while V0 did not. W1-W10 were washes collected from the Ni+2 agarose column using the breaking buffer whereas E1-E10 were washes collected using elution buffer. Figure 2. SDS-PAGE/Coomassie Blue stained gel analysis of rGFP SDS-PAGE gel, which was used to analyze our purification of rGFP from a crude extract, and the corresponding molecular weight. This gel was made with a 12% resolving gel and a 5% stacking gel. Wildtype rGFP has a molecular weight for 27kDa. The ladder has kDa rungs of 97.4, 66.2, 45.0, 31, 21.5, and 14.4. The band for rGFP has been indicated on the figure. The different samples were taken during our purification. The highest protein containing washes and elutions were used in the SDS-PAGE gel. G0 represents a sample containing rGFP prior to induction. G3 represents a sample containing rGFP after 3 hours of induction. GCE represents the GFP crude extract after the slow-freeze/quick-thaw process. Figure 3. Western Blot of rGFP We used the Western Blot to detect, and more importantly, quantify proteins that react with antibodies. The Ponceau S stain was performed to highlight the existence of our proteins successfully being transferred from the SDS-PAGE gel. We then performed a blocking step with 5% non-fat dry milk/TBS solution followed by washes with 0.05% Tween20/TBS. The probe first used was mouse IgG anti-Xpress epitope MAb solution, which we followed with washes of Tween20/TBS solution. The second probe used was sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution, which we followed with two washes of Tween 20/TBS solution with the third wash being pure TBS. Then, TMB solution was added to develop the nitrocellulose membrane and this reaction was stopped by pouring distilled water over the membrane. Above are the 8 lanes, G0, G3, GCE, W2, W3, E3, E4, and a known molecular weight ladder, respectively. CONCLUSION/DISCUSSION: A great amount of research has been put into the field of monitoring gene expression and there are currently many research laboratories that use labeled antibodies and other various means to do so. However, this new recombination technology, a vector can be created to provide a simpler and less expensive approach that can even be performed in vivo. The purpose of this experiment was fulfilled as His-6 tagged rGFP from a particular E. coli strain was successfully expressed, purified, qualified, and quantified by means of Ni+2 agarose chromatography. GFP was initially expressed as a histidine tagged protein that was inserted in E. coli and subsequently a crude extract of this protein was isolated. Throughout the experiment, we were able to inspect and measure the fluorescing activity of our elutions with the use of handheld UV lights. GFP was successfully expressed because of the fluorescent activity that was emitted when shown under UV light. This rGFP was purified using a Ni+2 agarose column which allowed for the binding of the His-6 tagged rGFP to the Ni+2 agarose beads in the column, and subsequently let all other proteins not of interest and containments to be collected in washes. Then, rGFP proteins were eluted by passing elution buffer containing Imidazole through the column because Imidazole has a higher affinity for the Ni+2 agarose beads than does Histidine. Therefore, one could expect that most if not all of the rGFP would be found in the elution washes and would present higher fluorescing activity than the washes with breaking buffer. Figure 1 clearly supports this argument. From referencing Figure 1, the most rGFP was found in E2 because it exhibited the highest fluorescent activity in RFUs. Using a standard curve based off of known quantities of mass of a certain protein, one is able to extrapolate the amount of protein present in ones sample. This can be used to estimate the amount of rGFP activity. For instance, E2s specific activity was found to be approximately 100,592.2 RFU/mg. W1-W6 had the least amount of fluorescing activity because the washes were done prior to the addition of Imidazole through the column. This is expected as Imidazole would flush out rGFP in the elutions, which would produce the highest GFP fluorescing activity. Percent purity of the rGFP samples were approximated using the SDS-PAGE gel with Coomassie blue staining to allow visible bands to appear which were used for comparisons. The molecular mass from each individual sample was determined by comparing the bands with the bands of the known weight ladder. The percent purity of each individual band was determined by comparing the bands against each other. The molecular weight of wild type rGFP is 27 kDa, and the molecular weight of rGFP for mutant rGFP used for this experiment was approximately 31.4 kDa with the 40 extra amino acids that were added taken into account. For example, E3 percentage of purity was found to be .4. The amount of protein yielded was 29.4 ug of rGFP for the third Elution fraction (E3) after a total protein amount of 49 ug The Western Blotting technique detected and quantified proteins that reacted with a specific antibody. In our case, it verified rGFP was expressed and whether or not it fell in the range of the expected molecular weight of 31.51 kDa. Lanes GCE, G0, G3, and E3 all contained a bright band around 31.4 kDa, indicating that rGFP was existent. This result corresponds to the expected of E3 having the one of the highest rGFP activity. The fact I had used E3 for the SDS-PAGE and Western Blotting skewed my results, but E2 and E3 had the highest activity for me. Even with my error, GFP was effectively expressed and purified and its finding is important to science because it can be used to track cancer causing cells one day to help figure out where problem spots begin and can help eliminate the issue before it escalates indicating that fluorescent proteins might eventually be clinically useful in cancer patients (Hoffman). This can occur since GFP can successfully be expressed in other organisms as this experiment through the use of E.coli, humans cells can certainly be tagged and label the tumour before treatment and then monitor for fluorescence after treatment to identify possible recurrence or metastases (Hoffman). The same can be said with heart disease and other health related problems. The future of GFP remains limitless when scientists can unlock its full medical potential. REFERENCE/BIBLIOGRAPHY (1) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Manual. The University of Texas at Dallas. Richardson, TX. (2) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Lecture Notes. The University of Texas at Dallas. Richardson, TX. (3) Ward, William. History of GFP and GFP Antibodies. Brighter Ideas. 2009. 26 Oct, 2010. http://www.brighterideasinc.com/proteins-antibodies/history-of-gfp-and-gfp-antibodies/ (4) Ninfa, Alexander J. and David P. Ballou (1998). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. (5) Hoffman, Robert M. Uses of Fluorescent Proteins to Visualize Cancer In Vivo: Can Fluorescent Proteins be Used in Humans? 2005. 28 Oct. 2010. http://www.medscape.com/viewarticle/513975_6

The Invisible Poor :: essays research papers

I believe that the wealthy Americans have moved farther away from the poor then in past generations. James Fallows in †The Invisible Poor† clearly shows how the new technology millionaires awareness of the poor has diminished greatly. I believe that this is due to several reasons the most important being the young age in which wealth and success are reached. The technology millionaires are much younger then previous generations of millionaires and thus are removed much earlier one. They find success very easy and can not see why others can’t obtain the same wealth as they have. They also limit their circle of friends to those with similar interests or backgrounds. They have limited time and only spend what free time they have (beyond family and work obligations) with those who they are comfortable with. This comfort is generated by their common threads such as education, similar employment or social activities. When the rich stay within this circle they no longer are exposed to the poor and thus they forget that they exist. When they are exposed to the poorer people it makes them uncomfortable (like when the cleaning lady was around) instead of dealing with it or helping they choose to avoid the situation. Figuring if you avoid it then you’re not admitting that it exists, thus not having to deal with them. I believe that since the article was written times have changed somewhat with the collapse of the stock market in a sense. Many people losing their jobs in the high tech world may bring some of them back to reality and acknowledge those less fortunate. They are now looking for jobs and finding that their skills were limited to the high tech industry. Many are now taking lower paying jobs to get by. They have discovered that the wealth of stock options are now worth allot less. Making many of them near poverty themselves.

Native Americans

For many years Native Americans life has been a struggle. The government at the time was trying to encourage them into the white world and ultimately lose their cultural beliefs but Indians themselves at the time fought bitterly against the policies by the US government and also for their tribal lands. Their major concern was that if they chose to live away from tribal land they experienced discrimination and if they lived in tribal land the US constitution did not apply to them. In 1961 tribes gathered for a meeting in Chicago where they issued declaration of Indian purpose that stressed responsibility of preserving their native land and the right to choose their own way of life. Due to the activism the congress passed the Indian Civil Rights Act of 1968 which granted Native Americans full access to the United States Bill of Rights and protection for their tribal lands. This means that the Native Americans can govern themselves in sovereignty. Even though civil right gave tribes legal rights and protection there were conflicts that arose between respecting Native American sovereignty and practicing the United States bill of rights. Robert, Ericson and D. Rebecca Snow, â€Å"The Indian Battle for Self-Determination†, 1970 this additional source was an article written by two scholars who explained the battle for self-determination for the Indians. The article provides new information about the controversy in American courtrooms and legal halls. The article stated, â€Å"Federal courts have traditionally viewed the tribe as a dependent or â€Å"tributary† nation possessed of limited elements of sovereignty and requiring federal protection;' the Congress has alternately viewed the tribe as a substantially independent political unit or as an anachronism which must be phased out consequent to a policy of Indian assimilation† (Erickson and Snow). This quote shows us that the tribal sovereignty has often been conflicted. Another important information I learned from the article was what a tribe cannot do when it comes to certain situations. The article claimed, â€Å"Moreover, a tribal Indian cannot claim protection from illegal search and seizure, or from interference with freedom of speech, press, or assembly. In addition, a tribe can impose a tax, or revoke tribal membership rights without complying with due process requirements† (Erickson and Snow). This quote explains the limitations of being a tribal Indian. This was an important information to learn because it showed the struggles of many Indians back then. Lastly, this quote explains one of the positive aspect of the civil rights act of the government. The quote states, â€Å"Perhaps the most encouraging aspect of the proposals is that they represent an attempt to respond to Indian grievances. This is a refreshing departure from past policy. Too often, Indian preference has been ignored† (Erickson and Snow). All of the examples provide new outside information about the Indian civil right Act of 1968. Finally, this source could be consider trustworthy and biased because even though it is written by a scholar but it is not like a newspaper from during that time of the event. I think that both of the authors agreed on the Indian civil right act but they states that there were conflicts and controversy in American courtrooms and legal halls. Both of the scholars liked how the government took an attempt to respond to the Indians because they often get ignored. The article was well balanced when providing information because not only it talked about the rights act but it also provide information about the government and how the Indian civil right act movement started. I think this was a good source to use because it gave me new insight about Indians and I learned more information about the civil right Act Itself.

Lord of the Flies: Intelligence and Good vs. Evil Essay

Good versus evil is a common concept used often in storytelling, writing, plays, movies, etc. the basic story line is commonly used and developed to mold different ideals, meanings, and lessons into different types of works. William Golding’s novel the Lord of the Flies falls into this category of works with the good versus evil story line. Boiling the novel down to its most basic state it is a story of a group of boys. They all start out in a state of innocence, then as they adjust to their new surroundings after being stranded on a deserted island with no adults; they chose whether or not they turn from their innocence. When the boys turn from their innocence they go from being good to evil, or as the interpretation of this novel is commonly perceived the boys go from being civilized boys to savages. It is in this sense that civilized and good can be used interchangeably for this interpretation, and the same for evil and savagery. Golding puts an interesting twist to this basic plot right in the beginning of the story. Golding takes the story from just a simple tale of good versus evil to good versus evil with competing ideas of intelligence. Right in the beginning of the novel we see these ideas of intelligence take form. As the story builds the differences in the types of intelligence grows and becomes more distinct. From there the competition of good versus evil begins. After their plane crashes the boys who were on the plane to escape the warfare in England are scattered on the island. Ralph, the first boy we are introduced to meets another boy named Piggy. â€Å"It’s a shell! I seen one like that before. On someone’s back wall. A conch he called it. He used to blow it and then his mum would come. It’s ever so valuable-â€Å"(15). With Piggy’s help Ralph uses the conch they found to call out to the other boys on the island. This is the first hint at the two types of intelligence. Piggy is already exhibiting signs of natural intelligence. This type of intelligence is developed based on the sensory analysis of the surroundings. This intelligence is more advanced and enables Piggy to think in more civilized, advanced ways. He is immediately made fun of for his appearance and as the story progresses is mocked as a know-it-all. No one listens to Piggy, even though the conch and the meeting were his ideas no one realizes this, nor do they care. Once all the boys are gathered together there is a vote on who should be chief. A boy named Jack is introduced as the leader of the choir boys and he wants to be chief, but when put to a vote Ralph is elected. Ralph does give the choir to Jack and asks what they would like to be. Jack tells Ralph that he and his choir shall be the hunters. Ralph depicts more social intelligence. Ralph knows how to work a crowd, how to lead a group, and how to gain respect. â€Å"Everybody must stay round here and wait and not go away. Three of us- if we take more we’d get all mixed, and lose each other- three of us will go on an expedition and find out† (23-24). This act showed that Ralph was able to get the attention of the boys at any time and that the boys would actually listen to him. His ability to be able to accomplish this as quickly as he did really shows his true social intelligence. â€Å"If a ship comes near the island they may not notice us. So we must make smoke on top of the mountain. We must make a fire† (38). Ralph does show some natural intelligence as well, but not nearly as much as Piggy exhibits. Jack also shows a form of social intelligence. He is able to keep command over his choir and they listen to him no matter what. This shows that in the beginning of the story even though the boys are all different, have different types of intelligence, and have mixed feelings about the situation they are still united together as a whole. â€Å"All day I’ve been working with Simon. No one else. They’re off bathing, or eating, or playing† (50). The boys begin to slack and begin to realize that there aren’t consequences or punishments for the wrongs they do. Jack and his group of hunters become obsessed with the idea of killing a pig, and are the only ones exempt from helping build shelters, though the other boys don’t really care enough about their orders to help build or gather food. It’s at this point in the novel where there are serious signs of cracks and issues with the order system the boys have put in place since being on the island. Jack is beginning to slip into a more savage state, and is using his social intelligence to bring other boys down with him. The improvised form of society that the boys have created is already starting to weaken and fall. â€Å"Kill the pig. Cut her throat. Spill her blood† (69). â€Å"Look! We’ve killed a pig-we stole up on them-we got into a circle-â€Å"(69). It is at this point in the novel where the group of hunters, the boys who killed the pig, have turned from innocence. The kill they had obsessed over finally happened and they were proud of themselves for killing. The act of killing a living creature, something they never had done before, was the true turning point for them. There was no turning back from it; the hunters even painted their faces, this sense of wearing a mask, pretending to be someone else enabled them to kill. â€Å"‘But they’ll be painted! You know how it is. ’ Eric says. The others nodded. They understood only too well the liberation into savagery that the concealing paint brought. ‘Well, we won’t be painted,’ said Ralph, ‘because we aren’t savages’† (Golding 172). Ralph is starting to understand that Jack and the group of boys who follow him are starting to turn to a more evil state. Ralph still tries to use his social intelligence and command over his boys so they don’t go over to Jack’s side. Within Jack’s group the obsession with hunting caused the boys to let the fire go out, and subsequently lose a chance at rescue. The hunters didn’t really seem to care about the fire; they were still overly hyped up about their kill, swearing that they would hunt again and bring back even more meat. As the boys become more and more content with letting go with the rules they get closer and closer to losing their sense of civilization completely. â€Å"‘If I blow the conch and they don’t come back; then we’ve had it. We shan’t keep the fire going. We’ll be like animals. We’ll never be rescued. ’â€Å" (Golding 92). As the boys start to fulfill their own wishes of hunting and playing they get sick of listening to Ralph and being controlled by the conch. They don’t want to do work; they just want to have fun. Jack rebels against Ralph and makes his own â€Å"tribe,† where he could be the leader and he would no longer be controlled by the conch. Evil starts to arise rapidly; the majority of the older boys side with Jack and abandon Ralph’s authority. After that Piggy, Sam and Eric are the older boys who remain with Ralph. As the night wears on, some of the â€Å"littleuns† sneak off to join Jack. This is truly the end of their makeshift society, it has completely failed. After the split of the group it appeared as though things could settle down, but Ralph and his boys find that it is nearly impossible to keep the rescue fire going. They need to ask the others for help but Jack’s boys are too afraid to help them. Jack had taken to using torturous and intimidation methods to keep control over his group. He wears a mask, like he did when he killed the first pig; this seems to allow Jack to give in completely to his â€Å"evil† savage side. It was while celebrating their savageness and reenacting the kill that they got carried away and got themselves overly worked up. â€Å"The beast struggled forward, broke the ring and fell over the steep edge of the rock to the sand by the water. At once the crowd surged after it poured down the rock, leapt on to the beast, screamed struck, bit, tore. There were no words, and no movements but the tearing of teeth and claws† (Golding 153). The wickedness in all of the boys deludes them into mistaking Simon, the only truly â€Å"good† pure boy, as the beastie. Simon was considered to be the â€Å"pure† boy, the epitome of civilization and innocence. Jack and the other boys had completely given themselves over to their evil sides, giving in to their natural instincts and savagery. Social Intelligence and natural intelligence are both qualities that help a person to be successful in life. However, overall, society puts greater worth in social intelligence. The abilities to work with others, lead and motivate others, and inspire others make a person successful in life. Just like good and evil, social and natural intelligence go hand in hand. One is not greater than the other, but like people believe good is better, people put more stock in social intelligence. In Lord of the Flies the boys put their trust in Ralph, but as time goes on, and rules begin to be broken, the less control Ralph seems to have. Social intelligence will only go so far, it can’t be natural instincts. Natural instincts are hidden behind the rules and expectations of society. When they begin to dwindle and those instincts become more prominent, that natural intelligence becomes important, it can be the intelligence or lack thereof that can make or break a situation. Without one, evil or good, natural intelligence or social intelligence, the other is not really known. Without evil how would we know what good is? If everyone was socially intelligent how would we know what natural intelligence is? The answer is we wouldn’t have a way of knowing. A person cannot know one without knowing of the other. Intelligence and good and evil all work together, as seen in Golding’s novel. Jack possessed the same kind of social intelligence as Ralph, but when he gave in to evil his social intelligence the power it gave him over the other boys allowed Jack to change the atmosphere of the island and turn the boys away from goodness and civilization. Piggy had natural intelligence but because he lacked any real social intelligence he was shunned, mocked, and in the end killed for this. He came off as a know-it-all and a whiner; this annoyed the other boys and eventually led to his demise. The boys didn’t put any stock in natural intelligence; they put all their stock in social intelligence and natural instinct. Ralph, with Piggy’s help, was able to remain civilized and good. Though he lost his hold on the boys he still had his social intelligence, and even some natural intelligence of his own to rely on. When Ralph was in charge his positive outlook affected the boys as well, they were positive because he was positive. The struggle between good and evil does not just affect the person who’s battling it within themselves, it also affects those around them, and subsequently can change them too. ?

The Definition of a Princess

Every little girl has once wished (upon a star) to one day be a princess in a fairy tale. However, do they truly understand what the definition of a fairy tale princess is? Fairy tales have created a definition and a set of requirements of a true princess, a definition and a set of requirements that could easily be applied to a pretty teacup or any other inanimate object. These fairy tales have defined a princess as someone who is unrealistically beautiful, passive, with no voice, and helpless and dependent. Beauty is one of the key requirements to be a princess. Every woman in a fairy tale is either a beautiful princess or an ugly witch or stepmother. There are no average looking women to be found in these tales. Since physical qualities are often an allegory to the goodness or evil of a character, beauty has been a requirement for a princess. In the picture books and movies of these fairy tales, audiences will see tall skinny girls whose waists are almost the size of their necks with eyes that are abnormally large. Though unrealistic, this appearance has been made necessary in these tales. Would Cinderella be let in to the ball or ever have a dance with a prince if she were ugly? Would Prince Charming even consider kissing â€Å"Sleeping Beauty† if she were â€Å"Sleeping Ugly†? Beauty is such an important part of a princess but how does it affect those young girls who wish to be like them? These fairy tales give the message that the only way to find love or Mr. Perfect is to be beautiful. Young girls watch and read storybooks and movies that have princesses with body types that are considered anorexic. This image will sink in to their minds subconsciously and affect them for the rest of their lives, since most children will never be able to reach the standard of beauty required to be a princess. Along with beauty, a princess has been defined to have no voice or say. In almost every fairy tale a princess never gets to express her opinion or personality. The ultimate example of this is in the Little Mermaid (Disney Version) when Ariel trades her voice so she can alter her appearance. She is told that she won’t need a voice to get the prince and all she needs to do is look pretty. This leads young girls to think their voice and opinion don’t matter and to have their â€Å"happily ever after,† all they have to do is stay quiet passive and be visually attractive. Lastly a princess is defined as someone who is helpless and dependent. Almost every single fairy tale has a damsel in distress waiting for the prince to save them. Sleeping Beauty has to wait for Prince Charming, Rapunzel has to wait in her tower, and Prince Eric saves Ariel. In all fairy tales this is a common factor; the princess is always helpless and needs saving. Never in a traditional fairy tale does a princess save the prince. In addition to the sexism in these tales, they present and promote a negative message for girls. It communicates to girls that if something bad is happening or if there is a problem, wait for a male to come and solve the situation. Putting these ideas in little girls’ heads at such an early age will discourage them from to ever becoming strong and confident women. Understanding the true definition of a fairy tale princess is, princesses should not be role models for so many girls. However so many little girls still want to be princesses, even if they are defined to be unrealistically beautiful, passive, voiceless, and helplessly dependent on others. This needs to change in order to have more confident and smart women. Society should think about these fairy tales and really consider if the princess in the tale is someone young girls should have as their role models.

Ethics Notes Chapters

Ethics, and Human Behavior Ethical issues exist in all areas of criminal Justice system (from passage of laws to punishment) Criminal Justice professionals have discretion Legislators: In making laws Police: In enforcing laws Attorneys and Judges: affecting Justice process Correctional Professionals: affecting offenders lives What do criminal Justice professionals have in common? Power to make decisions duty to enforce the law obligation to provide â€Å"due process† and â€Å"equal protection† for all commitment to â€Å"public service† Goals of the study of ethics -BrawledBecome aware of and open to ethical issues Begin developing critical thinking skills Become more personally responsible Understand coercive element of the Justice system Develop wholesales (the ability to explore with ones heart as well as ones mind) Defining Terms morals The judgment of behavior as right or wrong. Ethics The study and analysis tot what constitutes g * The two words are often used interchangeably. Or bad conduct. Meta-ethics Technical investigation of the meaning of ethical terms, as well as how ethical statements can be verified. Normative ethics Definition of right conduct and moral duties. Plied ethics Application of ethical principles to specific issues or fields. Professional ethics Examination of the behavior of certain professional groups. Duties Moral obligations that one must carry out to be considered ethical. Supererogatory An act that goes beyond duty and is not required to be considered DOD or moral. Imperfect duties General obligation with no specific acts. Honesty Health Family Financial success Beauty What else are values? Are some more important than others? To be Ouagadougou or immoral,behavior must involve: human acts of free will detecting others Inventory of Ethical IssuesIndividual or Other Employees backstabbing and lack of support gossip sexual or racial harassment lying to cover up blame taking credit for others' work Organizatio n and Employees sexual or racial harassment by supervisors discouraging honest criticism or feedback arbitrary or unfair decisions inadequate compensation inadequate training unrealistic or inappropriate demands putting employees in unnecessary danger By the Individual and Organization work ethic (days work for a days pay) petty theft of supplies or cash overtime abuse gifts and gratuities falsifying reports misuse of sick days arsenal use of supplies or equipment Morality and Behavior Even when people know what is right, sometimes they choose to do wrong?why? Criminology Psychology Other fields seek to answer this question Are people fundamentally good or fundamentally bad? Thinking Point Omar Thornton of Connecticut walked into his employer, Hartford Distributors, for a hearing concerning his possible termination. During the meeting, Thornton pulled out a pistol and shot ten of his coworkers, killing eight, before turning the gun on himself. What caused Thornton to carry oh t this minority act? Was he born intrinsically bad? Was he a good person that had a tragic turn of circumstances that forced him into a criminal act? Morality = Law ?Ethical Issues and Dilemmas Decentralization of soft drugs? Sex-offender registries? Death penalty? Mandatory DNA testing? Three-strikes legislation? Racial profiling? Steps for Clarifications Dilemmas Review all the facts. Identify relevant values of all parties. Identify all moral issues. Identify most immediate moral issue. Resolve the dilemma. CHAPTER 2: Determining Moral Behavior Ethical Systems Deontological Ethical Systems Teleological Ethical Systems Ethical Formalism According to German philosopher Emmanuel Kant: ill (motivation) is the only thing that is intrinsically good. Duty is required behavior. It is self-imposed and necessary to morality.The Categorical Imperative Ethical Formalism: Imperatives Categorical imperatives Are absolute. Are based on good will. Determine morality. You must not lie. Criticisms of Et hical Formalism Utilitarianism leered Beneath An action's morality depends on how much it contributes to the overall good of society. Humans are hedonistic. They seek to maximize pleasure and avoid pain. DOD for the greatest number On April 20th, 2010 a British Petroleum offshore rig exploded killing 11 employees ND causing one of the largest oil spills in modern history. Investigators soon located the faulty alarm systems. The alarms did not alert because they had been intentionally disconnected close to a year ago.BP had the alarms turned off in order to allow employees to sleep without being interrupted by false alarms thus creating a better functioning workforce. If the alarms were enabled, the rig would have automatically entered shut down mode, virtually eliminating the oil spill. Was Up's original â€Å"act† inherently good? Bad? Did they have a duty to act one way or the other? Criticisms of Utilitarianism Religion People hold different opinions about which religion i s the â€Å"true† religion. People within a religion often disagree on how to interpret its principles. Religious controversies are often difficult to resolve. Natural Law Criticisms of Natural Law How can we determine what is natural law versus man-made law? What are the â€Å"natural† laws of morality?The Ethics of Virtue Aristotle True virtue is the median between extremes of character: the golden mean. People develop moral virtues through practice, Just like any other strength. Daemonic: living the â€Å"good life† Six Pillars Josephs Institute) Trustworthiness : Honesty, sincerity, loyalty Respect : Golden Rule Responsibility : Being accountable Fairness : Equality, impartiality, and due process Caring : Altruism and benevolence Citizenship : Duties of citizenship Criticisms of Ethics of Virtue Basically assumes a good person will make a good decision. Little help for people facing dilemmas. The Ethics of Care Western ethical systems focus on issues like ri ghts, laws, and universalism.Ethics of care?nurture, meeting needs Criticisms of Ethics of Care Criticisms of Egoism Violates universalism. Logically inconsistent (for everyone to try and maximize self interest). Enlightened egoism is not too different from categorical imperative or golden rule. Other Methods epithetical Decision Making The imperative principle directs a decision maker to act according to a specific, unbending rule. The utilitarian principle determines the ethics of conduct by the DOD or bad consequences of the action. The generalization principle is based on this question: â€Å"What would happen if all similar persons acted this way under similar circumstances? General Principles federation Making Obtain all facts (including the effects of prospective decisions). Evaluate whether odd be comfortable with your decision appearing on the front page (in public view). Consider one's principles to be like a formula – applicable in all situations. Relativism Ethic al Relativism Moral systems are products of an individual or group. If people believe different things are good and bad, how can you define what is good? Situational Ethics: A compromise between relativism and absolutism There are basic principles of right and wrong. They can be applied to ethical dilemmas and moral issues. They may call for different results in different situations. 1.Treat each person with the utmost respect and care. . Do one's duty or duties in such a way that one does not violate the first principle. Summary of Ethical Systems Ethical formalism: What is good is that which conforms to the categorical imperative. Utilitarianism: What is good is that which results in the greatest utility for the greatest number. Religion: What is good is that which contorts to God's will. Natural law: What is good is that which is natural. Ethics of virtue: What is good is that which conforms to the Golden Mean. Ethics of care: What is good is that which meets the needs of those c oncerned. Egoism: What is good is that which benefits me.

The Blending of Art and Mass Culture Essay Example | Topics and Well Written Essays - 1500 words

The Blending of Art and Mass Culture - Essay Example While he asserted that "the poster isa mass-produced object that must have a commercial function" (Medlej), Cassandre found that he could use images, colors, and text in a sort of artistic fusion to grab the attention of the viewer. Making posters as large as 13' x 15', Cassandre learned how lucrative art could be (Owen). As Neil Harris observed, "business [could] beas stimulating a patron of the arts as prelates, cardinals, and popes were who represented the church in the fifteenth century." Cassandre's approach was not without its detractors. There were those who wondered if his style would make its way from France into American advertising - specifically, whether the American advertising designers would be willing to set aside their more conservative and monotonous design styles for something that seemed to swing closer to the world of art than the world of business. Those who hoped that American advertisers would change their designs were those who saw how Cassandre's posters used geometry to grab the attention of the viewer; they realized the potential of "attention value." While advertising only attracts those whose "circumstances permit the possibility of action," Cassandre sought to make sure that the most focused attention would be on his posters (Parker). This focus would come with the elimination of distractions, so that the central message of the advertisement would be clear to the viewer (Parker). Cassandre established a handful of principles in his poster design. As mentioned above, Cassandre aimed for very large posters, and had a revolutionary attitude toward text. While other poster-makers attempted to squeeze the text in around designs, or wherever it would fit, Cassandre found the text to be the central part of the poster, particularly in advertisements. He claimed that "design should be based on the text and not inversely" (Medlej). His emphasis on the artistic value of text led him to experiment widely in sans serif fonts (Megaw, Hatchings), even to invent the Peignot-font, which he believed to be a "purer form of the alphabet" as far as it expressed the "essential character of Roman letters" (Tam). His goal was to make Peignot one of the established text-faces, but this did not come about during his lifetime (Tam). Cassandre's emphasis on text even went to the use of alliterative slogans to increase the allure to the viewer (Hambourg). Another one of his principles centerd around the use of geometry and architecture to design his posters. Enlarging a poster to the sizes that Cassandre wanted in the 1920's risked severe distortion of the original image, and so lines were used that would be difficult to distort. This use of line and form in a more figurative way was one of the basic influences in the later works of Max Lingner and Salvador Dali, who took the distortion of line and form to new frontiers (Held). Cassandre's notions of poster-making had a significant effect on a variety of art forms throughout the rest of the twentieth century. His ideas concerning the use of line and color to increase audience attention became one of the founding tenets of the Bauhaus school.