Construction of Recombinant DNA in E Coli

Construction of Recombinant DNA in E Coli In 1973 Stanley Cohen and Herbert Boyer pioneered the use of recombinant DNA technology for cloning and expression of genes in foreign organisms. They cloned DNA from the Salmonella typhimurium streptomycin resistance plasmid RSF1010 into the Escherichia coli plasmid pSC101 and observed tolerance to streptomycin among the transformants (Cohen et al., 1973). The first reported production of a human recombinant protein took place a few years later when the then newly started biotech company Genentech announced that they had managed to express the gene encoding human somatostatin in E. coli (Itakura et al., 1977). The value of the resulting bioactive substance was similar to that of somatostatin extracted from the brains of 500.000 sheep. In 1982 Genentech followed up this success with the product humulin, a recombinant insulin produced in E.coli and the first recombinant biotech drug to be accepted for market by the Food and Drug Administration. Today the production of recombinant prot eins has become a huge global industry with an annual market volume exceeding $50 billion (Schmidt, 2004). At the start of the recombinant protein expression era the bacteria Escherichia coli and Bacillus spp. dominated as hosts for recombinant expression, but the realization that a protein may require a specific host physiology and biochemistry for optimal production stimulated a search for new hosts, both prokaryotic and eukaryotic. Parallel to this quest, recombinant DNA technology advanced tremendously thereby opening up possibilities for the use of novel organisms. As a consequence, many different expression systems for use in many different hosts are now available, including systems for use in yeasts (Gellissen et al., 2005), filamentous fungi (Nevalainen et al., 2005), insect and animal cell cultures (Wurm, 2004; Kost et al., 2005), gram-positive bacteria like Bacillus (Westers et al., 2004) and Streptomyces (Binnie et al., 1997), and gram-negative bacteria like Escherichia c oli Bacterial expression systems are the preferred choice for production of many prokaryotic and eukaryotic proteins. The reasons for this lie in the cost-effectiveness of bacteria, their well-characterized genetics, and the availability of many different bacterial expression systems. Among the hosts available for recombinant expression, Escherichia coli is in an exceptional position. This stems from the many decades of intense researchon its genetics as well as the broad scope of biotechnological tools available for genetic engineering of this organism. As a host for recombinant expression, E.coli is especially valued because of its rapid growth rate, capacity for continuous fermentation, low media costs and achievable high expression levels (Yin et al., 2007). One consequence of this popularity is that about 80% of all proteins used to solve three-dimensional structures submitted to the protein data bank (PDB) in 2003 were prepared in E.coli (SÃ ¸rensen and Mortensen, 2005) and during 2003 and 2006, nine out of 31 approved therapeutic proteins were produced in E.coli (Walsh, 2006), among them important growth factors, insulins and interferons (Schmidt, 2004). Green Fluorescent Protein (GFP) was isolated from the jellyfish Aequorea aequorea in 1962 (Shimomura et al., 1962) where it was found as a companion protein to aequorin, the well-known chemiluminescent protein of the same species. It was noticed that living A. aequorea tissue had an emission spectrum peaking at 508nm and looking green but pure aequorin peaked in the blue range, at 470nm (Tsien, 1998). This then led Shimomuras group to discover GFP and suggest radiation-less energy transfer as the mechanism for exciting the protein. Its structure has been determined to consist of an 11 stranded ÃŽÂ ²-barrel containing the chromophore made up of a single ÃŽÂ ± helix as shown in Figure1. Its use as a tool in molecular biology was not realised until 1992 when Prasher reported the cloning and sequence of GFP (Prasher et al., 1992). Since 1994 GFP has been used as a reporter protein (Chalfie et al., 1994) flagging its own presence and therefore also proteins under the same control, by emitting green light (ÃŽÂ »em = 508 nm) upon excitation with near ultraviolet light (around 395 nm) or blue light (around 470 nm) (Ito et al, 1999). Since then many mutations have been developed looking to improve the emission or to focus it to a single wavelength (Heim et al., 1995) or to change the color of the emitted light itself. Recombinant DNA molecules usually contain a DNA fragment inserted into a bacterial vector. Polymerase chain reaction (PCR), a specific gene or DNA region of interest is isolated and amplified by DNA polymerase extracted from a heat-tolerant bacteria. PCR finds the DNA region of interest (called the target DNA) by the complementary binding of specific short primers to the ends of that sequence. The long chromosome-size DNA molecules of genomic DNA must be cut into fragments of a much smaller size before they can be inserted into a vector. Most cutting is done with the use of bacterial restriction enzymes. These enzymes cut at specific DNA sequences, called restriction sites, and this property is one of the key features that make restriction enzymes suitable for DNA manipulation. These enzymes are examples of endonucleases that cleave a phosphodiester bond (Anthony, 2012). The key property of some restriction enzymes is that they make sticky ends. The restriction enzyme EcoRI (from E.coli) recognizes the following sequence of six nucleotide pairs in the DNA of any organism: 5-GAATTC-3 3-CTTAAG-5 The enzyme EcoRI makes cuts only between the G and the A nucleotides on each strand of the palindrome (Figure.2). The recombinant DNA molecules are transferred into bacterial cells, and, generally, only one recombinant molecule is taken up by each cell. The recombinant molecule is amplified along with the vector during the division of the bacterial cell. This process results in a clone of identical cells, each containing the recombinant DNA molecule, and so this technique of amplification is called DNA cloning. The next stage is to find the rare clone containing the DNA of interest. Bacterial plasmids (vectors) are small circular DNA molecules that replicate their DNA independent of the bacterial chromosome. The plasmids routinely used as vectors carry a gene for drug resistance and a gene to distinguish plasmids with and without DNA inserts. These drug-resistance genes provide a convenient way to select for bacterial cells transformed by plasmids: those cells still alive after exposure to the drug must carry the plasmid vectors. However, not all the plasmids in these transformed cells will contain DNA inserts. For this reason, it is desirable to be able to identify bacterial colonies with plasmids containing DNA inserts. Such a feature is part of the pUC18 (or pUC19) plasmid vector shown in Figure 2; DNA inserts disrupt a gene (lacZ) in the plasmid that encodes an enzyme (-galactosidase) necessary to cleave a compound added to the agar (X-gal) so that it produces a blue pigment. Thus, the colonies that contain the plasmids with the DNA insert will be white rath er than blue (they cannot cleave X-gal because they do not produce -galactosidase). The following experiment outlines the construction of recombinant protein production in E.coli strain BL21 by using a bacterial plasmid vector pUC18/19 expressing Green Fluorescent Protein (GFP) to act as a recombinant protein product with the benefits of being easy to visualise and measure. Materials and Methods Materials: The experiment was carried out using the following materials and Equipments: 2Â µl EcoRI/HindIII cut and cleaned PUC19 vector, 5Â µl EcoRI/HindIII cut and cleaned GFP insert, 2Â µl 10xT4 ligase buffer, 2Â µl T4 ligase(0.5 U ml-1) , and 9Â µl sterile water (H2O) ]to make up to 20Â µl volume[ . 100Â µl of competent BL21 E.coli cells on ice, 42Â °C water bath, Ice bucket with ice, selective media plates (1.5% Luria broth (LB) Agar, 40Â µg mL-1 X-gal, .1 mM IPTG, 50Â µg mL-1 ampicillin), sterile tubes, shaking incubator, Spectrophotometer or similar device to measure optical density of the bacterial cultures, flasks, Microcentrifuge. Methods: It can be divided into three stages: Ligation Reaction stage: in this stage 2Â µl EcoRI/HindIII cut and cleaned PUC19 vector, 5Â µl EcoRI/HindIII cut and cleaned GFP insert, 2Â µl 10xT4 ligase buffer, 2Â µl T4 ligase (0.5 U ml-1) , and 9Â µl sterile water (H2O) are mixed and kept at room temperature for at least 30 minutes. Transformation of ligation into cloning host stage: this stage conducted by deforesting 100Â µl of competent BL21 E.coli cells on ice (with caution do not allow to warm to room temperature), then adding 10Â µl of the ligation reaction from the first stage to BL21 E.coli cells. They are then incubated for up to 30 minutes on ice. Next step, is done by taking out the transformation mixture out of the ice and heated in water bath at 42 Â °C for almost 75 seconds, then followed by return immediately into ice for a minimum of 2 mins. Then the cells were plated out on selective media plates (1.5% Luria broth (LB) Agar, 40Â µg mL-1 X-gal, .1 mM IPTG, 50Â µg mL-1 ampicillin). Lastly, the transformation mixture is incubated at 37 Â °C for 12-18 hours afterdriedd. Picking of colonies for the protein expression stage: 2x5ml LB +50Â µg ml-1 ampicillin in 30ml sterile tubes were prepared, then 1xBlue individual colony and 1x white individual colony selected and inoculated in separate tubes. Then the tubes were incubated with shaking incubator throughout the night at 37Â °C , speed: 220rpm. Subculture and Growth of Recombinant E.coli for Protein expression: At the beginning, 2x60ml sterile Luria-Bertani (LB), in 250ml conical flask were warmed , (1 per inoculums ) at 37 Â °C, Then aseptically the ampicillin was added to a last concentration of 50Â µg ml-1 ampicillin. Next 1 ml of media was removed and was put in a cuvette to act as blank (one blank is enough for both ouh), followed by addition of 600Â µl overnight to calture of each individual colony to separate flask (1:100 inoculum), the flasks were put back to the shaking incubator and incubated at 37Â °C, speed: 200rpm , after that blank spectrophotometer was placed against media at 600nm , after 45 minutes the samples were removed aseptically from flasks, then from every flask 1x 1mL was removed and added to a fresh clean cuvette (take to next step 8) and 1x1ml was added to clean Eppendrof (take to step 9) . The OD600nm of culture in cuvette was Measured and the result of growth curve was recorded (once the cul ture has reached an OD 600nm of 0.5, IPTG was added to final concentration 1Mm stock solution. Then samples were spun down in the Eeppendrof tube at max speed in Microcentrifuge for 5 minutes , ensure centrifuge is balanced before spinning , the supernatant was removed and pellet ,then the pellet was suspended in 200Â µl Cell lysis buffer (10mMl Tris PH8.0, 300Mm Nacl , 10mg ml-1 Lysozyme). Resuspended cells were frozen at -20 c to the next day. Lastly, sampling was continued until OD600nm is no longer rising for two successive samples or until 16:30 pm. Results and discussion Although it is supposed to harvest between 30-300 colonies per plate (210- 2100 colonies for all groups), just three blue colonies were observed in plates between all groups, which mean that protein of interest (GFP protein ) was not expressed (inefficient) in BL21 E.coli cells due to some factors influenced the expression level or to some technical problems during the experiment which will be discussed. The most popular strain, BL21 and its derivatives, which are good producing protein, are descended from E.coli B and thus is deficient in the Lon protease. Additionally, the BL21 background lacks the OmpT outer membrane protease. For expression work, BL21 cells should be taken from stock cultures that performed from fresh transforms. This step is crucial to insure that the clone does not change and that each expression run gives optimal performance. Transformation frequency is affected by the purity of the DNA, how the cells are handled, and how the transformation was actually performed. In the impurities in the DNA usually spin columns can be used to purify DNA from PCR reactions, ligations, endonuclease digestions, or other treatments. In addition, the most common mistake when transforming E.coli is to put a lot of ligation mixture in the transformation. Other factors that effect transformation with BL21 are the handling and the storage of the competent cells. Competent cells need to be reserved at -70Â °C to keep them at the peak .It is worthy of noting that 5-10-fold of efficiency usually lost if tube put back in the box and place in the freezer. Moreover, Cells must be thawed on ice, and the transformation should be started immediately after the cells are thawed. Incubating on ice is necessary for chemically competent cells. If you heat shock right away, the efficiencies will be down 10-fold. If incubate for only 15 minutes, it will be down 3-fold. In addition, time of heat shock (75 second) could be not enough , thus, affect the efficiency enough to transformation of E.coli. Moreover, water bath temperature may be not equilibrated (less than 42Â °C or a higher which decrease in transformation efficiency ( Smith, et al, 1992). Also, the concentration of DNA has significant effect on the transformation efficiency , usually less amount of DNA is used. If using more, the result is fewer colonies because the impurities in the DNA will inhibit some of the cells from being transformed. There are main factors to consider during induction conditions: Vector, Host Strain, and Growth Conditions. These three factors have the biggest impact on the expression of the protein of interest. First on the list of considerations is the vector that is used to express GFP protein. The first thing should be considered after cloning, the protein of interest is still in frame. It is recommended that before any experiment is carried out the first thing is should be done is cloned plasmid (or a few different clones) sequenced. This will show if the sequence you inserted into the expression vector is still correct and is still in frame. This is especially important if the construct contains any PCR fragments. If there are any point mutations or the sequence gets out of frame by even a few bases it can have dramatic effects on the protein that expressed. Another thing to check before expressing is if the GFP protein sequence contains long stretches of rare codons. This can cause the prot ein that is expressed to be truncated or non-functional. A few rare cordons spread around the protein are OK in most cases, but if there are a number of rare codons in a row, then it can have a big effect. The third sequence related step to optimize the protein production is to make sure there is not a high GC concentration at the 5 end of GFP protein. This could potentially cause problems with the mRNAs stability, and could prevent it from being translated correctly, which would also lead to truncated or non-functional proteins. If your sequence is GC heavy at this end, you can try to make a few silent mutations to break up long stretches to try and help stability. After the plasmid is sequence verified, the next factor is the bacterial host that is used. There are almost as may hosts as there are expression vectors, with certain hosts excelling in producing different types of proteins. For example if you have a toxic protein, or a protein that could potentially cause genomic rearrangement, you will want a vector that gives you very tight control over the induction of your protein. There can be leaky expression (i.e. expression of your protein without the addition of your inducer) that can potentially have adverse effects on the cells growth or even prevent your cells from over-expressing your protein in the first place. If youre utilizing the T7 polymerase system, then look for a host containing the pLysS plasmid, as this will code for T7 lysozyme, which will suppress the T7 polymerase and can greatly reduce the level of background expression. If as stated before you have a protein that contains a large number of rare codons, then look for a h ost with the genes for the necessary tRNAs already present, which should allow your protein to express correctly. Sometimes simply changing hosts can have a dramatic effect on the amount of protein produced and the stability of the protein that is made, so if one host isnt giving you the results you need, then feel free to switch your host up. The third and final factor to consider when expressing a protein is growth conditions. When first starting out with the protein induction it is very important to run an expression time course, where you take a fresh colony from a streaked plate, and grow the culture to stationary phase. Next, dilute the overnight culture 1/100 and grow to mid log phase, then add the inducer and induce your protein for a number of hours, taking 1mL samples every hour or so. Once these samples are lysed, you can run an SDS-PAGE gel to determine your protein production levels. You might get great induction the first time, or you may have to tweak your conditions in order to get really good expression levels. Other factors that may need to be controlled for are the bacterial growth rate (determined by taking OD measurements during the induction process), and the temperature during induction. Some constructs will express perfectly fine at 37Â °C, while others need to be bumped down to 30Â °C to induce c orrectly. The concentration of the inducer too will have an effect, as many inducers (IPTG) can be toxic to the cells that they are inducing. Using freshly made inducer is good step to making sure you always have consistent results. Only through experimentation can you determine what will be best for your construct, and give you the most robust expression levels. Transformation efficiency: Transformation efficiency is a measure of the ability of cells to be transformed. Transformation efficiency is expressed as the number of transforms per microgram of pUC19. By using the following formula: Colonies on plate / ng of control DNA X 1000ng/Â µg = (transformation (T) / Â µg plasmid DNA) 100 ÃŽÂ ¼L equivalent to 0.01 ng DNA in the plate. Growth curve In general growth curve shows the S- shaped when plotted in log linear format as shown in figure 4, that separated into four phases: Lag phase; the initial period when no increase in cell number is seen. Log phase; when cells are growing at the maximumm rate. Stationary phase; growth decreases as a nutrient are depleted and waste products accumulate. Death phase; this is the result of prolonged starvation and toxicity. Conclusion The main goal for the experiment was to express the protein of interest (GFP). However, factors influencing transformation efficiency include technique errors, the temperature and length of the incubation period, the growth stage of the cells, and using the correct mass of plasmid DNA. Escherichia coli is one of the most important hosts in modern day recombinant protein production. Throughout academia and industry its uses are widespread and with sequence data available for some of the most common strains of the bacteria it has been a favourite organism for many metabolic engineering and metabolic modelling projects in the past (Berry, 1996; Koffas et al., 1999).

Federalism and Separation of Power

Democracy as a system of political administration has been termed over years as a product of several institutions working together to ensure the sustainability of an exclusive political system. Democratic institutions in a state are saddled with the responsibility of sustaining a nation’s democratic process such institutions like legislature, judiciary and the executive are the major institutions that guarantee efficiency in a democratic system. ( Ologbenla 1996) Basically, the legislative arm is the major institution that guarantees such because it represents the generality of the people’s interest.The doctrines of separation of power and checks and balances are two major tenets of democracy. Both doctrines provide basic principles that should be upheld in any democratic state. The adherence to these democratic tenets depends largely on the level of political adherence that follows the basic rules and regulations that guides the conduct of both the ruler and the ruled in an exclusive political community. Such rules and regulation are codified in a document known as constitution. A constitution is a legal framework that spells out the composition, function, and jurisdiction of government officials. Almond et al. 1966) It is a body of fundamental rules guiding the affairs of state. It states the relationship between the governors and the governed. Separation of powers is a doctrine propounded by Baron de' Montesquieu which stipulates that in order to avoid arbitrary use of power, power should be decentralised and shared among the organs of government such that no organ becomes too powerful. (Neumann 1949) The principle of checks and balances states that an organ of government should act as a watchdog on the other organs of government so as to curb their excesses.In a democratic system all governmental powers are derived from the constitution, it also spells out the functions and relationship of major governmental institutions such as the executive, judiciary and the legislature such that no organ of government can interfere in the affairs of the other. The constitution makes each arm of government to be an independent and coordinate unit, independent in terms of its sphere of influence and coordinate in its inter-governmental relationship with other arms of government.With cognisance to the American democratic structure, the constitution provides for separation of powers by stipulating the functions of the various arms of government and also the jurisdiction of the different tiers of government, whereby the executive cannot meddle in the affairs of the legislature and vice-versa. The primary function of the legislature is the making of laws ;( Easton 1961) it would be a total negation of the principle of separation of power if such function is being exercised by the executive.Although, the executive can propose a bill after much deliberation by the legislature can be passed into law but the power to make laws lie in within th e jurisdiction of the legislature. But in recent times the principle had been challenged due to the overwhelming power and personality of the executive. For example, President George Bush after the Sept 11 attack on the world trade centre proposed a bill to the congress to invade Iraq. Before the house could pass the bill he had sent troops to wage war in Iraq.This was contrary to the constitutional provisions which states that before United State would engage in a war it must be ratified by the congress in a joint session. Checks and balances on the other is to serve as a balance between the various organs of government in such a way that an arm of government serve as the watchdog over the other arms of government. (Neumann 1949) This function is majorly that of the judiciary this is the done through judicial reviews which help scrutinize both activities of the executive and the legislature.For this function to be performed to the optimum level there is the need for an independent judiciary that is free from executive manipulation. A bill is a proposed law that is not yet law until it is passed by the law making body in the country and received the executive or presidential assent. (Easton 1961) There are several stages that are involved in the passing of a bill before it becomes law. The first stage of the bill is the first introduction of the bill to the house. The introduction of bill could either be a private member bill or it originates from the lower house depending on the type of legislative chamber in operation.In a two chamber legislative house, bills originate from the lower house and are deliberated on in a joint session. This stage marks the first reading of the bill to the house. The second reading marks a stage where the bill is fully deliberated upon by lawmakers and it represents a crucial stage in the passing of such bill into law, because this stage determines whether such bill would become law. After the bill had successfully passed through the second reading then a committee would be constituted to critically examine and analyse the bill, give recommendations and possible impact of the bill if passed into law.This stage represents the committee stage. After constituting the committee, the next stage is the report stage where the committee presents their report to the house on the bill. After the committee stage the bill is presented to the lawmakers for adoption. It should be noted that at this stage the bill can still be rejected if the lawmakers refuse to adopt the bill by voting against it. But if the bill was accepted by the lawmakers then it can now proceed to the third stage which requires the president’s assent.If the bill passed by the legislature was not assented to by the executive, the legislature can constitutionally veto such bill into law after a period of 14 days. Federalism is a political system in which governmental powers are shared among the different tiers and organs of government such that each tier and organ is coordinate, independent, and exclusive in its own sphere of authority. (Leslie 1954) With reference to the debate on whether state power had been reduced or increased in a federal structure, cognisance would be given to emerging democracies mostly in third world countries where democratic structures are still growing.In Nigeria, state powers are gradually reduced as the constitution vested much power in the exclusive legislative list which only allows the federal government to legislate. (Ologbenla 1996) Matters such as currency, defence, health, mining, state creation, local government creation, boundary adjustment, leaving the state with little area to exercise its sphere of control. Unlike other federal structures like the United State of America where states have the autonomy on state police, the Nigerian federal structure did not provide for such provisions even at the agitation of states to have their autonomy on the issue.In 2003, the Lagos state gover nment embarked on the creation of local government which was later regarded as unconstitutional and led to a legal matter between the state and the federal government. (Tadese 2012) The judgement was later passed in favour of the federal citing that states do not have the constitutional right to create such establishment. In the American federal structure allows for state power to be shared between the central, state, and municipal governments in such a way that each state has its own constitution where it derives it powers from.Although when such laws clashes with national constitution the latter prevails. Federalism has been the major factor sustaining the democratic values as it has it functionality in both the principle of separation of powers and checks and balances which is maintained through the efficacy of institutions that guarantees a smooth democratic process.References Almond Gabriel, Gabber Powell. (1966). Comparative Politics: A developmental approach; Little Brown ; C o, Boston. Print. Easton David. (1961). A framework of political analysis, Yale University Press, New Haven. Print. Leslie Lipson. (1954). The Great Issues of Politics (5th edition) Prentice-Hall, New Jersey. Print. Neuman Franz. (1949). Introduction to Montesquieu’s Spirit of Laws. Translated by Thomas Nugent: Halfner publishers, Chicago. Print. Ologbenla Derin. (1996). Introduction to political science, Olucity Press Limited, Lagos. Print. Tadese. Oyeniyi. A battle of legal supremacy; Lagos State faces FG on creation of local government. Vanguard Newspaper. Web 30 September 2012.

Mirror Imagery in The Scarlet Letter by Nathaniel Hawthorne Essay

According to research in the field of psychology, guilt manifests itself in many ways. Often those who feel guilty see assurances for their feeling in the action of others—even when the public has no interest in his or her private life. In a conservative society, however, rules are imposed upon him or her, barring the person from moving ahead with their life, no matter how insignificant the crime. Michael L. Lasser takes a similar approach, arguing that Pearl is a mirror image of Hesters guilt—a constant reminder of her mistake. Lassers argument has merit because Hawthorne not only uses mirror imagery in relation to Hesters guilt, but also in regards the emotions of all characters. In Mirror Imagery in The Scarlet Letter, Michael Lasser argues that Hawthorne uses mirror imagery to reveal a characters innermost secrets and ulterior motives. The child Pearl is described as, the scarlet letter in another form; the scarlet letter endowed with life! Having made this statement, he explains his argument through Hesters impressions of Pearls eyes—full of smiling malice. When Hester and Pearl visit the governors mansion, Hester notices a look of naughty merriment in the little girls eyes. Hawthorne also mentions a fiend that occasionally peeps out of Pearls eyes. Through his description of Hesters impression of the evil that lurks within Pearl, Lasser comments on the notion that Pearl is evil by using the Puritan statement that no good comes from evil. Since Pearl is illegitimate and the result of an act of sin—the ultimate Puritan evil—she is seen as evil as well. Lasser explains further that Pearl is not only the embodiment of Hesters sin, but also of her conscience. Lasser explains that Pearl knows her mothers deepest feelings in a way uncommon to a child of her age. Thus, Lasser illustrates that Pearl is used a symbol—a mirror— for Hesters guilt. Lasser argues that Hawthorne uses similar mirror imagery techniques with Dimmesdale and Chillingworth as he does with Hester. In his final moments, Dimmesdale holds a private vigil by his mirror in which he sees diabolical shapes—representing Dimmesdales untold sin—angels, and finally Hester and Pearl. The description the soul beheld its features in the mirror of the passing moment compares time to a mirror and the soul contemplating its past as the image that appears on the mirror. On the other hand, Chillingworth is  a reflection of his own malevolence. His warped body represents his inner ill will—his desire to torture Dimmesdale. Hawthorne further describes Chillingworths eyes, saying, Sometimes a light glimmered out of the physician’s eyes, burning blue and ominous, like the reflection of a furnace, or, let us say, like one of those gleams of ghastly fire that darted from Bunyan’s awful doorway in the hillside, and quivered on the pilgrimâ₠¬â„¢s face. Chillingworths eyes are used as a reflection of his evil—the ghastly fire that lives within him. Lasser concludes that Hawthorne uses such imagery to imply certain views upon a reader, leaving the reader to carry this suggestion throughout the story. If we look closely at the reactions Hester sees in her child, we can see the characteristics of a guilt-ridden mind. People are constantly judging me and their judgment is important to me is how someone psychologically bothered by his or her guilt would think. This perception of continuous judgment is very obvious with Hesters reaction seeing even her child demonstrating accusations in various forms. A child does not know how to be polite and socially appropriate when speaking. Such habits come from years of training. Often maturity is judged by the tolerance one develops towards others mistakes. Even her normal reaction of trying to play with her own reflection in water is described with reference to a mirror. The picturesque detail found in the sentence Here and there, she came to a full stop, and peeped curiously into a pool, left by the retiring tide as a mirror for Pearl to see her face in ties in the mirror as one of the objects connected with the story in readers mind. The brook itself is a mirror of Pearl. Hawthorne describes the brook as, [Gushing] from a well-spring as mysterious, and had flowed through scenes shadowed as heavily with gloom. Pearl, like the brook, springs from an unknown source—her mysterious parentage—and flows through a world filled with gloom and guilt. In addition to making explicit references to mirror and reflections of images, there are many instances where mirror is implied. For example, whenever Hester suffers Pearls playful acts, it is her inner turmoil that is mirrored in the acts of the child. Hester views her child as the product of a crime and, therefore, an evil entity; this is no surprise knowing the  ideas that existed in a puritanical society wherein they saw the child as an extension of his or her parents characteristics. Thus the societys ideas are reflected and perpetuated by even those who are victims. Since such behavior has not been eradicated even in the current, modern society, it is only natural to expect a puritanical society to have brainwashed Hester to feel guilt towards her childs actions. One day, as her mother stooped over the cradle, the infant’s eyes had been caught by the glimmering of the gold embroidery about the letter; and, putting up her little hand, she grasped at it, smiling, not doubtfully, but with a decided gleam that gave her face the look of a much older child. Then, gasping for breath, did Hester Prynne clutch the fatal token, instinctively endeavoring to tear it away; so infinite was the torture inflicted by the intelligent touch of Pearl’s baby-hand. By describing Pearls intuitive grasp of Hesters guilt—her letter A— Hawthorne enforces Pearls role as a mirror of Hesters conscience. My imagination was a tarnished mirror, says Nathaniel Hawthorne in his introduction to Scarlet Letter. Michael Lasser picks out such references throughout and shows us the writers mastery in making mirrors an important symbolic artifact, be it the shiny breastplate that magnifies the letter A on Hesters chest or the brook that reflects shadowy and intangible qualities of the characters of this story. Bibliography â€Å"Mirror Imagery in ‘The Scarlet Letter'† Michael L. Lasser, The English Journal, Vol. 56, No. 2 (Feb., 1967), pp.274-277: National Council of Teachers of English, http://www.jstor.org/stable/811696

Effects Of The Reconstruction Era - 1139 Words

Effects of the Reconstruction Era The end of the Civil War created many short term and long term effects. After the Civil War, 1863-1896, United States, the north and south are trying to reunite by Rebuilding the Nation, to become unified and avoid being attacked by other countries. Through 1896, the North and South tried to reunite to avoid being vulnerable from attacks by other countries. The government tried to solve key problems after the war with Rebuilding the Nation. This was called the Reconstruction Era, their solutions were short term and failed to address the problem. Disagreements over Reconstruction also led to conflict in the government and in throughout the South, these disagreements led plans to solve the problem,†¦show more content†¦States could then be readmitted once 10% of voters had sworn allegiance to the Union and that state had declared to end slavery. This plan was short term in result to Lincolns assassination. Johnson had tried to follow through w ith the plan, but it did not work out. Another plan was made by the Radical Republicans who wanted to punish the southerners. They believed that the main goal of Reconstruction should be a total restructuring of society to guarantee blacks true equality. The bill, said that anyone who voluntarily fought for the Confederacy could not vote. States could be readmitted once 50% of all voters sworn allegiance to the Union and the state had declared to end slavery. After Rebuilding the Nation with short term plans, the States continued battling over the Reconstruction. Disagreements over Reconstruction led to conflict in government and in the south. Johnson decided to come up with a plan of his own. His lenient plan said that the south could become citizens if they swore to the United states. Johnson then became impeached due to breaking the Tenure in Office Act. In result of this, Johnsons lenient plan became a short term solution. Then the 13th, 14th, and 15th amendments were passed by congress in an attempt to resolve conflicts in the nation. The thirteenth amendment was passed to abolish slavery. The fourteenth amendment was passed, which addresses rights and equal protection of laws and was proposed to issues related to slavesShow MoreRelatedEconomic, Social, and Political Effects of the Reconstruction Era798 Words   |  4 PagesPrompt: What were the long-term economic, social, and political effects of Reconstruction? The United States was challenged with many issues after the Civil War like crop lien work contracts, segregation, and unresolved problems with the seceded states. This period was called Reconstruction. After the Civil War, African Americans were free but with no place to live in or to work at, they settled with their former ‘masters’. African Americans were technically free, but no one wanted to hire a coloredRead MoreThe Reconstruction Era and Its Effects on Slavery with and After President Lincoln2128 Words   |  9 PagesThe Reconstruction Era and its effects on Slavery with and after President Lincoln The Reconstruction Era which followed the Civil War was a period marked by a severe effort to re-establish a depleted and distraught society. The war, which was aimed at confronting the national dilemma of slavery, only led to subsequent problems over emancipation and an undefined condition of freedom. Some, who had naively assumed that ending slavery would resolve the problem of racial inequality, overlooked theRead MoreThe Reconstruction Era And The Jim Crow Era1525 Words   |  7 PagesThe Reconstruction Era and The Jim Crow Era were both times of Rapid growth in the United States that were characterized by changes not only on the intrapersonal level, but also on the cultural and legislative level. 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On December of 1863 the decree of â€Å"soft plan† was introduced. The â€Å"soft plan† included amnesty for the southerners that took the loyalty oath. It alsoRead MoreHow Did The Radical Republican s Rise For The Failure Of The Post Civil War Reconstruction?1619 Words   |  7 Pageswar reconstruction? The time between 1863, when Lincoln passed the ten percent act, until the year 1877, when reconstruction was officially ended, will be evaluated with information provided by the sources. The investigation will specifically look to how the Lincoln assassination allowed for the rise in the Radical Republican Party from 1866 to 1868 and the party’s effect on reconstruction acts leading to the failure of the post-civil war reconstruction era. Eric Foner’s novel Reconstruction: AmericaRead MoreThe Civil War Was A Grave Cause Of Many Events. Many People1663 Words   |  7 PagesThe civil war was a grave cause of many events. 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William Golding s Lord Of The Flies - 1513 Words

â€Å"Civilization begins with order, grows with liberty, and dies with chaos.† -Will Durant. This is demonstrated when a new war begins, and a group of boys are left to fend for themselves on an abandoned island due to a plane crash. They must learn to live without adults, rules, or structure, however, as time goes on and conflict grows, this becomes increasingly difficult. William Golding s Lord of The Flies explains how without order, unity, and rules a society will resort to chaos/savagery and becomes useless, as shown through the character archetypes of Learner, Ralph, and Shadow, Jack. The Learner character archetype, who begins innocent/simple, changes and overcomes obstacles, and gains experience/knowledge, is shown in Ralph as he†¦show more content†¦I’d like to put on war-paint and be a savage. But we must keep the fire burning. The fire’s the most important thing on the island, because, because-’ [Ralph] paused again and the silence bec ame full of doubt and wonder† (199). Ralph now has gained some experience and learned that fire is the most important thing and realizes the savagery the island withholds. He still tries to keep hold of unity and civilization with his meetings and jobs, however, due to the conflict with Jack and their opposing views, Ralph has trouble staying grounded, forgetting why they even need the fire and losing sight of his leadership and motives. The civilization suffers from this, and the savagery slowly makes it’s way into the remaining boys. As time went on eventually all boys, except for Ralph, had joined Jack’s group of savages, and they attempt to hunt Ralph as though he were merely a pig. They smoke him out and Ralph runs towards the beach, wounded, for safety. A naval officer sees the smoke and comes to rescue them, stopping the boy’s only seconds before Ralph would have been killed. Once the boys explain that they were separated and were having a war, as w ell as that Ralph is their chief, and that at least two boys were killed, they begin to lose their composure. The naval officer turns his back as â€Å"Ralph wept for the end of innocence, the darkness of man’s heart, and the fall through the air of the true, wise friend calledShow MoreRelatedWilliam Golding s Lord Of The Flies1263 Words   |  6 PagesResearch Paper: Lord of the Flies William Golding, the author of Lord of The Flies, included adults for only a brief time throughout the novel, playing only a minor role at the end. The absence of adults exemplifies how children require the structure and guidance that only parents can provide, symbolically, how nations newly freed from the British Empire’s control would be better off under English colonial power to survive and maintain order before deteriorating into anarchy. The adults of theRead MoreWilliam Golding s Lord Of The Flies752 Words   |  4 Pagespossible, so a five year old who teases others to Adolf Hitler would be classified as perpetrators of evil. Lord of the Flies is a fictional story about a group of British boys who get stranded on island. The author of the novel Lord of the Flies, William Golding, showcases Zimbardo’s ideas in his story. Zimbardo did not form his theory Through the character development of Jack and Roger, Golding illustrates the intensity of evil when one is impacted by situational forces. Before Ralph and Piggy unifiesRead MoreWilliam Golding s Lord Of The Flies1869 Words   |  8 PagesEssay Outline – Unit 11 Introductory paragraph: Topic Sentence (includes the book title and author) The novel Lord of the flies by William Golding is a type of literature that revolves around an anti-war theme. Main Points that will be discussed in the essay presented in order of weakest to strongest: 1. Lord of the flies was written during WWII and one of the manifestations is the dead man in the parachute presumably a victim of a bombed plane. 2. Faction among the group which is similar toRead MoreWilliam Golding s Lord Of The Flies1282 Words   |  6 PagesWilliam Golding, the author of Lord of The Flies, included adults for only a brief time throughout the novel, playing only a minor role at the end. The absence of adults exemplifies how children require the structure and guidance that only parents can provide, this can be seen how nations newly freed from the British Empire’s control would be better off under English colonial power to survive and maintain order before deteriorating into anarchy. The adults of the novel can be seen as the motherRead MoreWilliam Golding s Lord Of The Flies1389 Words   |  6 PagesA response to Lord Of The Flies Imagine an airplane crash. The heat of flames scorch passengers’ backs in addition to the wind burning their faces. Lucky, this crash was over water and near an island so most passengers survive, with an exception of the airplane staff and the pilot. Even though alive, many are in fits of fear and panic, and others are in shock. After hurried deliberation, a lone member of the group is elected leader in hopes that they will calm the panic, and make the hard, but necessaryRead MoreWilliam Golding s Lord Of The Flies1315 Words   |  6 PagesWilliam Golding’s novel Lord of the Flies was set somewhere on the timeline of World War Two, a war between the Axis and the Allies lasting from 1939 until 1945. Although WWII was fought between many countries in the Pacific and Europe, the main contender was Germany, led by Adolf Hitler. Hitler and his followers, the Nazis, changed the lives of everyone when they attempted to strengthen Germany and brought out all the evil and ugli ness in the world. After WWII, nothing would be able to change theRead MoreWilliam Golding s Lord Of The Flies886 Words   |  4 Pageshow to live their lives not knowing what s right or wrong. Everyone has a different opinion towards different things. Some say gun laws should be banned while some say they want a gun in their house. In Lord of the Flies by William Golding there are clear aspects of leadership shown within the characterization between Jack and Ralph. I m chief, said Ralph, because you chose me. And we were going to keep the fire going. Now you run after food- (Golding 150). There is evident conflict between theRead MoreWilliam Golding s Lord Of The Flies1672 Words   |  7 Pages The Different Social Cognition of the Similar Stories — Synthesis essay of Lord of the Flies Final Project With the development of British culture, the format of Desert Island Literature has an inevitable connection with the geographical and culture heritage of the development of British history. Generally speaking, the setting of such literature is basically around an isolated island which is far from human society. The characters usually follow a primary lifestyle so that illustrate the courageRead MoreWilliam Golding s Lord Of The Flies1745 Words   |  7 Pages1954 novel, Lord of the Flies by Nobel Prize-winner William Golding is a dystopian allegory indicative of vast aspects of the human condition. Set in the midst of a nuclear war, the text details a group of marooned British school boys as they regress to a primitive state. Free from the rules and structures of civilisation and society, the boys split into factions - some attempting to maintain order and achieve common goals; others seeking anarchy and violence. The novel is based on Golding’s experienceRead MoreWilliam Golding s Lord Of The Flies1776 Words   |  8 PagesMaybe the beast is us (Golding 85), in the novel, Lord of the Flies, by author William Golding, Golding uses the entire book as social commentary. The social aspect he focuses on is man’s ability to be evil and destructive. William Golding uses three specific literary devices to convey this idea; characterization, diction and symbolism. Lord of the Flies explains man s capacity for evil which is revealed in his inherent human nature, which he cannot control or ignore. The hidden evil within

Essay on Customer Empowerment - 3312 Words

Customer Empowerment The Choice is Yours The Internet has permanently changed the relationship between consumers and the retail industry. Electronic commerce has provided consumers with more options, more alternatives and more opportunities than ever before. Consumers are no longer limited to physically visiting main street or big-box retailers. Instead, they are able to choose from products and services from companies large and small, located all over the world, without leaving their homes. Tangible points of comparison between retailers, which now can be automatically aggregated by software buying agents in seconds, include more than selection and price. Shipping costs, return policies, privacy practices and†¦show more content†¦The customer has always been right, but its never been like this. At the same time, these same customers are learning more about their choices and the legal aspects of privacy protection. During the next 12 months, retailers must combine airtight privacy policies with business models that defer to customer empowerment. Those businesses that do not place customer service above all else will fail. Online retailers face three primary challenges. First, they must attract customers by rising above the competition through costly marketing and promotional campaigns. Second, they must compel customers to regularly visit the site through a quality user interface and overall shopping experience. Third, they must lock the customers in all the way through the checkout. In an environment where the next competitor is a mouse click away, failure to overcome any of these three challenges could be detrimental. Even with a huge potential market, attracting customers to a site is expensive. With per-customer acquisition costs ranging from $29 for Amazon.com to more than $250 for some online brokerages,ii keeping customers is as important as attracting them. These high costs underscore the importance of a traditional retail adage -- the best customer is the one you already have. Long-term relationships are based on qualityShow MoreRelatedCustomer Satisfaction, Loyalty, and Empowerment1282 Words   |  6 PagesMarketing Dr. Arni Arnthorsson Customer Satisfaction, Loyalty, and Empowerment 1. Should a company be happy or concerned if most customers are satisfied? Satisfaction of the consumer is one of the main goals for a company to achieve. Customer satisfaction is defined as the feeling that results when an offering meets a consumer’s expectations. Companies around the world are doing many surveys, in order to measure the satisfaction of their consumers because the importance of consumer satisfactionRead MoreHow Employee Empowerment Is The Most Important Resource For Any Company1516 Words   |  7 Pagescompany will have an impact on the overall company’s performance. Empowerment refers to a process in which a manager shares power with a subordinate. The dictionary’s definition of empowerment is to give someone the authority to do something. Employee empowerment is the process of enabling an employee to think, behave, act, react and control their work in more autonomous ways as to be in control of one’s destiny. Employee empowerment is a motivational technique that is designed to improve performanceRead MoreThe s Balance Between Culture, Benefits, And Boundaries Essay1517 Words   |  7 Pages Information on this helpful website expanded instantly and the corporation became a collectively Internet source. Above all, the corporation moment of fame came when it received its million-hit in 1994, interpreting to thousands of diverse customers. Yahoo! advanced so expeditiously that the pioneers of this website thought there were some promising business favorable circumstances in this website. This essay will discuss organizational structure, the balance between culture, benefits,Read MoreThe Grand Regency Hotel Management Essay1634 Words   |  7 Pagesmanagement roles. The strategies included: empowerment of the employees; reduction of rules that led to bureaucratic behaviors; enhancing innovativeness of the employees and increased interaction between the management and the employees. All these were new strategies that had never been used before at the hotel hence employees needed time to adapt. Analysis of the Empowerment Strategy One of the aspects that he always used in his management duties was empowerment of his employees whom he trusted andRead MoreEmpowerment - advantages and disadvantages1198 Words   |  5 Pagesempower is defined as to give official authority or legal power to therefore employee empowerment would be giving official authority and power to employees, or giving the employees responsibility for what they do. Employee empowerment, or just empowerment, has many positive and also many negative or controversial sides, throughout this paper I will discuss these points and discuss them in depth. Empowerment is a trend that has been hitting companies since the 1990 s. The movement had originallyRead MoreThe Performance Challenges Of Electra Products Success Essay1651 Words   |  7 PagesElectra Products success and to provide recommendations for workplace development. The insight this report provides is limited by the information supplied on Electra Products which pertained mainly to the monthly meeting introducing a â€Å"new era† of empowerment. Assumptions have therefore been made for the purposes of completeness. Giving consideration to the history of downsizing it is assumed that the organisation is pursuing a no-growth to low-growth business model (Cameron, 1994) while looking to improveRead MoreEmployee Empowermen1 Essay1198 Words   |  5 Pagesï » ¿ Employee Empowerment: Organizational Achievement Through Employee Engagement Management of Human Resources MGT-331-CL02 Ronnie Ramirez April 20, 2014 Dr. Kathryn Adamson, PhD. TABLE OF CONTENTS I. Introduction 2 II. Issues Addressed 2-4 III. Analysis of Issues Read MoreEvaluating Safety Culture And Sms Implementation1399 Words   |  6 Pagesassess the safety culture currently present in that organization. What one can do to assess whether or not a company promotes a positive safety culture is to look at 6 critical components: organizational commitment, managerial involvement, employee empowerment, accountability system, communication and a reporting system. A successful company with a positive safety culture will consist of the majority of the aforementioned components. Without these elements, a company’s safety culture will appear non-existentRead MoreThe Rights Of Service Delivery1123 Words   |  5 Pagesdemand and/or improve services is effective because individuals at the local level know their own realities and are able to provide the best solutions. Rights based approach to development is based under the premise of facilitating a process of empowerment to citizens, and through collective action, hold the Government accountable to honor their human rights and legal entitlements. â€Å"Adopting a rights-based approach†¦ involves not only focusing on the content, but also on the process through which theseRead MoreCorporate Culture : Improving The Workplace1640 Words   |  7 Pageswhat motivates them and keeps them happy. Job satisfaction is related to performance, motivation, turnover, and organizational commitment. Empowerment stems from job satisfaction, and can be defined as â€Å"a set of motivational cognitions shaped by a work environment and reflect the individual’s active orientation in their work role.† (Psychological Empowerment and Job Satisfaction, Wang, Guangping) The components that result in an empowered worker are meaning, competence, choice, and impact. Meaning

Critical analysis fight club movie Essay Example For Students

Critical analysis fight club movie Essay An example of this is in one of the beginning shots which uses the CGI animation and his narration to inform the viewer about the explosives and project mayhem which are yet to be explained. The narration in Fight Club is vital in pushing the film further on in terms of plot, because without it the audience would not have the required knowledge to fully comprehend what is happening. Throughout the film, CGI is heavily depended on to show things in a particular way, for example showing the explosives by going from a shot of Pitt and Norton through the buildings is another enhancement to add style, which has allowed Fight Club to be known as a greatly recognised film. Lighting and colour are also an important issue to analyse when trying to evaluate the meaning of a shot. A good example of the use of colour is in the sixth shot that was analysed in the breakdown with the CGI animation. Throughout the animation the filter effect changes, giving the shot a different coloured tint. This is noticeable when the shot changes from being outside where the shot has a dark blue/ grey tint to inside when the colour changes to a lighter yellow/ orange tint. Colour is useful to give more meaning to a shot, in this example the lighter yellow/ orange tint represents safety whereas all the shots inside with the explosives and in the room the two characters are, represent a danger which again can lead the audience to wander why the room where the two characters are is dangerous. Throughout the film, the only two filters used are the blue/ grey filter and the yellow/ orange filter; this is useful for viewers watching to help sustain a meaning throughout the film, not confusing them. Elements of style work more unconsciously, meaning is hidden and the interpretation deciphers and translates. (Gibbs Pye, 2005; 216) It is up to the viewer to figure out the meanings behind what is happening on the screen, it is also up to them whether or not they want to. Fight Club has a lot of meanings behind each aspect of what is shown on the screen to help enhance the film as a whole. The interpretations that can be taken away simply need to be viewed with an analytical eye to see how very dark the film can be at the same time attempting to address the issues raised with a lighter feel. I believe that the 10 shots that were analysed in the shot breakdown together are there to introduce the film by showing viewers part of the end of the story so that they will need to find out how it begins and ends using simple mise-en-scene alongside computer animation to impress. Bibliography Gibbs. J Pye. D (2005), Style and Meaning, Manchester, Manchester University press Dudley. A (1984) Concepts in film theory, Oxford, Oxford University press Fight Club, (1999), USA Show preview only The above preview is unformatted text This student written piece of work is one of many that can be found in our University Degree Fine Art, Design Studies, Art History, Crafts section.