Emerson And Feudalism Essays (811 words) - Feudalism,

Emerson And Feudalism ?America was opened after the feudal mischief was spent, and so the people made a good start.? Was Ralph Waldo Emerson correct in that assertion? Why or why not? ? How were a person's rights and responsibilities determined in the feudal era? How are a person's rights and responsibilities determined in the United States today? ? What evidence is there in the U.S. Constitution that Americans rejected or accepted beliefs that were commonly held in the feudal era? To begin to fully understand what Emerson really meant in his speech from Boston's Old South Church, we must break it down. First, when Emerson speaks of the feudal mischief being spent, he means that the peak of the feudal era has passed in Europe when the colonists began to arrive in North America. In the second half Emerson asserts that because the colonists came over to America to build their own governments, without the influence of feudalism. These governments, free of feudalism, were an indication to Emerson that the colonists had made ?a good start.? While Emerson was right in the assertion that America made a start free of feudalism in the early stages of the colonies, he was incorrect in assuming that America would never experience ?feudal mischief? again. A resurgence of feudalism by King George in later years caused the colonists to revolt. Until 1763, the colonies existed in a state of salutary neglect, where the King and Parliament relatively ignored the colonies, allowing self-government. After 1763, England decided to enforce a policy of mercantilism, in which the mother country protected the colonies in exchange for exclusive trading rights. New legislation like the Stamp, Tea, and Quartering Acts eerily reminded the colonists of feudalism because their rights were being limited and they didn't have a voice in their government. As Adam Sandler said, ?Man does not know he is free until he has been in chains.? But the resemblance to feudalism in the later colonial era did not end with the revolution. In fact, there is evidence that aspects of feudalism survived in the United States Constitution. When the colonists came to America, they created a radically different government than the one they left behind in England. Though feudalism had waned by the 12th century, England was still left with vestiges of the feudal system, such as the rigid class system. In America, the colonists were allowed to build their own society from a state of nature, destroying the societal barriers that existed in their homeland. However, the ?feudal mischief? of which Emerson speaks was not over when ak similar to the vassal to lord relationship of the feudal system. When the colonies were founded, they operated under a mercantilist policy with England. Under this policy, all raw materials and resources of the colonies went without question to England, where they were manufactured into goods and sent back to the colonies. The colonists had no representation in England, and England had supreme power over the colonies and also had the duty of protecting those colonies from attack. This is nearly identical to the feudal system. However, England abandoned the practice of feudalism during the reign of King George, who followed a policy of salutary neglect. During this time the colonies were left to govern themselves. Then with the Proclamation of 1763, the king reasserted his feudal power over the colonists by restricting them to the land east of the Appalachian Mountains. This was followed by other laws that took away the powers that the colonists were used to having, such as the Intolerable Acts and the suspension of habeas corpus. This resurgence of feudal power caused the colonists, who had been practicing self-government, to rebel against England. Therefore, in a way the existence of feudalism caused America to be its own nation. The problem of feudalism was that it took away too many of the colonists rights. In the feudal system, the rights and responsibilities of the peasants and lords are clearly laid out. Similar to the early mercantilist system of the colonies, the peasants in a feudal system are required to serve their lord in a militia, and they are required to pay tribute to their lord. In England, this tribute was usually paid with the

buy custom The Legalization of Marijuana essay

buy custom The Legalization of Marijuana essay Introduction Marijuana is a substance with a very rich history. Its use dates back so many years ago. It is first referred as a psychoactive agent in 2737 BC in the writings of a Chinese emperor. During this period, marijuana was basically used for various medical purposes. Some of the medical problems whose solution lay in marijuana include; rheumatism, gout, malaria and absent mindedness. These were all effectively managed by this substance. In India however, marijuana found its use in recreation as it was loved by Muslims since consumption of alcohol is prohibited by the Koran. It was from India that it eventually spread to most of the regions of the world. 1545 is the year that marijuana eventually made its way to America. Introduction of this substance in United States is credited to the Spanish who ferried it to the New World. The English were later to introduce it to Jamestown in 1611. This substance was grown as a source of fiber. By 1890, the use of marijuana as a source of fiber had been replaced by other products like cotton. At this period, marijuana was already a constituent of many drugs used for medication purposes. Prohibition of marijuana was later to popularize it in 1920s. Jazz musicians and people involved in show business are some of the groups that found pleasure in recreational use of marijuana. This was later to spread out and joints sprang up that sold the substance mainly for recreational purposes. It is however understood that between 1937 and 1947, the American Government spent a lot of money; approximately $ 220 billion to paint this substance as the root cause of evil in the society and hence declared its use illegal (marijuana.com). It is in this respect that this research paper asserts that the war against marijuana is not issue based and marijuana should therefore be legalized. Decongestion of the prisons Time has come for us to ask ourselves what is best for us; whether it is the continued congestion of our prisons by marijuana users or hard core criminals. All researches in the recent past on the crimes committed by prisoners in different prisons in the entire nation point towards the direction of marijuana (Marijuana.com, 2011). It is important for all American to be open-minded throughout this crucial national debate. Our criminal justice system is not even moving closer to winning the war against recreational marijuana consumption. The war has a very long history and there is nothing to show for it. There are so many criminals in the society that are supposed to be pursued and locked down in our prison facilities. This is not happening because the facilities are filled up by marijuana offenders. Congesting the prisons by marijuana offenders does not help improve the image of our legal system. It is very important for us to understand the painful fact that the prisons do not have any positive impact on the inmates. Marijuana offenders while in prison only serve their incarceration and thereafter win their freedom back to the streets where they continue smoking their recreational substance as if nothing ever happened (Sasha, 2010). Even more discouraging with locking down marijuana smokers in prisons is the unhidden truth that marijuana still makes its way to the prisons and therefore the so called marijuana law offenders do not pose in their consumption of this drug. If at all anything changes, it is the cost of accessing it but not curbing its use. In order to make maximum use of our prisons, there is a need for legalization of marijuana not only for medical purposes but also for recreation where it is widely used by members of the society (Ruschmann, 2004). This way, our police will be able to go for hard criminals instead of hanging around to arrest the marijuana smoker as this has grown to become a bother to the society and not a solution to the consumption of the drug as it is supposed to be. Our prisons have a better role to play than this monotonous game that has by far and large lost purpose (Douglas Marneffe, 2005). National Economy It is fascinating when one takes time to go through the amount of money that is channeled to our Criminal Justice System in the fight against marijuana consumption. Billions are spent in each state to curb the use of this substance. There are so many problems that can be financed by this budgetary allocation on the fight against marijuana. Think about the fight against obesity, this is a serious problem in the society; does it get funding that is comparable to the amount spent in the fight against marijuana? The answer is a very strong NO; an indication that something is supposed to be done, attention is given to the wrong issue and there I a need for this to be corrected (Douglas Marneffe, 2005). There is an important aspect of the socio-economic growth and development that the anti-legalization of marijuana campaigners fails to recognize; the fact that the so many members of the society serving prison sentences as a result of breaking laws forbidding marijuana consumption have a role to play in the growth of the economy. So many people are of the impression that marijuana users are social misfits, people without any contribution in the economic growth. However, the truth is different; doctors, teachers, engineers, architectures, accountants, to mention but a few are all rotting in our prison facilities. We should be asking ourselves if these people were not performing their professional duties or the consumption of marijuana impacted negatively on their discharge of duties prior to their arrest and sentence (Sasha, 2010). The reality of the matter differs with this thought totally; there is no reflection of these thoughts with what is seen. There are so many professionals who are perfectly discharging their duties while using marijuana; the only difference between themand their incarcerated peers is the fact that whereas they are still free to contribute towards economic growth while their counterparts in the prisons have been denied a chance to do so. The war on marijuana therefore not only robs the economy of its already gathered resources by directly channeling what it has to the Criminal Justice System but also locking out able men and women from making their contribution towards the building of a strong economy. There is therefore a need for legalization of marijuana in order to allow these people to continue making their contribution to the economy and hence feel more appreciated. It is a degradation of their economic capacity by locking them in the prisons thinking that they will leave those facilities better man and women when the truth is different. These are normal ordinary men and women whose services are an asset to the society and the economy; let us not convert them to liabilities bearing in mind the big names that have made their way to prisons due to marijuana smoking. Legalizing marijuana will greatly boost the economy through increased tax revenue collection. A research carried out in the state of California revealed that tax from a $ 14 billion industry went uncollected annually due to failure to legalize recreation marijuana consumption (Inciardi, 1999). The same research went ahead to explain that 300,000 citizens of this state were already users of medical marijuana. This gives a vivid picture of what the country looses in tax due to its failure to legalize marijuana. The economy therefore stands to benefit a lot from legalization of marijuana as it is the case with cigarettes and alcohol. Once marijuana is legalized, legislations will be formulated that will give a guideline on how the respective states will be able to earn revenue from this trade. It is true that many fear that it would be complicated to collect tax from this trade. This is however in essence one of the many fears surrounding marijuana and would be demystified once the legi slation that legalizes marijuana in the country becomes enacted. The fact that other drugs like tobacco and alcohol are legal and tax revenue is earned from them is enough evidence that support from all free minded citizens is what is needed in order to have the wheel rolling and hence see our economy growing strongly by tapping this money that ends up in individuals hands. It no longer time to continue with the kind of hypocrisy that constitutes the fight against legalization of marijuana. Tax revenue from marijuana will be as important as is that from any other industry. We have to accept the truth, our economy has traces of money from marijuana and what is needed is to have a framework that will enable us to cut the roots of criminals who have illegally been benefiting from the trade without paying tax. Failure to earn this revenue as long as the industry is still illegal makes the government to be the looser in this game. The revenue collected from this industry is what should be used to take care of the victims of drug abuse. Even with mariju ana as illegal as it is, the government expenditure on the victims of drug abuse is high, yet the fight itself no matter how strong it is does not at any point move closer to success (Pudney, 2010) Corruption The fight against marijuana has always benefited few individuals in the society. Marijuana traffickers, just like other drug dealers are well connected people in the society. This is the main reason for the lack of success in the war. As we continue with the fight, we create more wealthy individuals in the society through this trade. It no secret that money from marijuana, just like other illicit drugs goes a long way in funding election campaigns in the country (Douglas Marneffe, 2005). This is the main reason for this scrupulous leaders continued campaign against legalization of marijuana. Their interest in the anti-marijuana campaign is strongly embodied in their personal interest; they want to ensure the industry is illegal so that they can continue to blackmail the drug barons into giving them money. Time has come for able men and women to realize the need to embrace legalization of this industry to end this form of corruption in our state offices (Mares, 2009). Our Criminal Justice System is another corruption overwhelmed sector that benefits from illegalization of marijuana. The police and other law enforcers all earn money through fraudulent means and always support the anti-marijuana legalization campaign. Do you ever take your time to find out how marijuana makes its way to our correctional facilities? Corruption is usually the way to go and it can only be fought by addressing the source; we should not continue to pretend that marijuana is bad when indeed those sending such messages rely on them in order to earn an illegal income through corruption (Mirion Zwibel, 1995). It is therefore vital that we close all avenues through which corruption retards our ability to formulate effective policies. It is only this way that we shall say we are in the right direction towards the fight against the negative effects of marijuana. We must therefore legalize the drug and then focus our efforts on addressing the negative effects that a few individ uals may show as a result of the use of the drug. We can always learn from the situation in Netherland in our attempt to better our policies regarding marijuana consumption (Pudney, 2010). Regulation Legalization of marijuana will pave way to formulation of key legislations that will be crucial in the effective management of the drug (Inciardi, 1999). Critical legislations will give the definition of the minimum age required for one to be able to use this drug. This will be important in protection of the rights of all members of the society. Just pose to think about the role of children in the illegal marijuana trade. The picture painted is such a worrying one. Minors are used in pushing the drug. However, once the drug is legalized, it will mean that there are no more secret dealings which are central in employment of minors in pushing the drug. Legalized use of the drug will give the definition of where the drug can be purchased, whether in operating drug stores or new ones specifically meant for marijuana. This way, one will be able to access the drug with a lot of ease without having to put the life of other citizens at risk. Another important of regulation is that the rights of those who do not use the substance will be respected. This way, smoking zones will be defined by the legislation and therefore a user will not only know where to go for the drug but also where and when to use it. This way, many lives will be saved as illegal access to the drug and consumption usually put so many people at risk. Fights that often erupt in the secret marijuana bases will be there no more. These fights occur quite often and are a reason for speedy measures to be taken in order to protect the involved citizens. As mentioned earlier, an American citizen is a valued asset of the state and his/ her life should not be put at risk whatsoever the case. It is the declaration of the drug as illegal that goes further to create all the social crimes that we witness. Let there be regulations to guide the trade and all will be well (Gerber, 2004). We have had fights erupting in restaurants and other social places, what is usually important is addressing the aftermath and putting measures to curb resurgence of the same. This is what we need to see even in the marijuana industry and not the simplistic argument that marijuana is the root cause of uncountable social problems. The society will always play host to social misfits and this should not be used to deny good citizens their access to what gives them happiness. Danger from violent black market criminals Leaving marijuana in the hands of violent black market criminals is creating insecurity in the country. No one can estimate the cost of what these dangerous criminals are capable of. For how long are we going to sit back and watch as young men and women are hacked to death by these criminals? The people dying are innocent people, only trying to kill their hunger for the drug but due to tight legislations that outlaw the use of marijuana, they are forced to use fraudulent means to get it. This lands them in the hand of these dangerous criminals who do not have any respect or value for human life. To them, it is simple; you either cooperate or lose your life. So many young people are being wasted by these drug pushers since once you get an orientation, they do not let you go away after the purchase, they expect you to create a new for them to a new market, that is network for them. This is the major route through which marijuana makes its way to schools. The only protection that the so ciety can get is legalization of marijuana so that these forced involvements into the use and later marketing of the drug will be countered (Pudsey, 2010). Acceptance in medication So far, 15 states have legalized what they call medical marijuana. These states include: Alaska, Arizona, California, Colorado, Hawaii, Michigan, and Washington State, New Mexico among others. This is a positive move towards legalization of marijuana for recreation as well. It must be pointed that the figures for medical marijuana users has been growing significantly in the states where it is legal (marijuana.com, 2011). It is also proven that the support for marijuana legalization has risen by over 25% since 1995 (Sasha, 2010). This is an indication that the people have come to accept the reality, that is, marijuana is there to stay forever and not a mere passerby. It is this support that Pro 19 lacked at the time of its introduction and hence was defeated. The growing awareness on the need to legalize marijuana speaks of itself. People are tired of living a life of denial and illusion. Concerned parents are no longer willing to hide their inability to stop their sons and daughters from smoking pot and instead want a shift in the manner in which they do it. Their security is now what they are interested in and of course are ready to invest in. They want the situation to be no different with that taking the drug for medication; that are free to march to licensed drug stores, present their cards and have the drug sold to them (Pudney, 2010). Market segmentation Once marijuana is legalized, dealers will be able to own licenses that will enable them to legally carry out marijuana business. These people will therefore do their best to safeguard their licenses since involvement in any other kind of drug business other than marijuana will see them arrested and their licenses revoked. This will therefore help in reduction of pushing of other drugs like cocaine, heroin among others that are illegal (Gerber, 2004). Conclusion Debate on the legalization of marijuana has often received mixed reactions from pro-legalization campaigners and the anti-legalization groups. Majority of those in support of legalization are marijuana users with the support of prospective farmers. This group believes that they stand to benefit a lot from a free environment as opposed to the current one that pushes them to carry out their activities in great secrets and when discovered, they have to spend their time behind bars for a considerable period of time. The anti-legalization campaign team is composed of the conservatives who question the moral virtues in the use of marijuana. It is however evident that the society will benefit more once marijuana is legalized when compared to the woes that define the illegal environment in which this business is practiced. 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Summary Essay Example | Topics and Well Written Essays - 500 words - 100

Summary - Essay Example Through censoring hostile traditional ideology was stack to the past giving room for civilization and modernization. Addie Prater Graham birth is estimated to be before 1900 in eastern Kentucky Mountains. She grew up around music and at the end of it she herself was a master in singing. Addie’s songs recorded in 1970s by her grandson Rich Kirby and Barbara Edwards, reflect as far back as the British Isles alongside others created in America as ditties, frolic songs and religious songs in line with the vintage Baptist tradition. Despite it being against the Baptist tradition to use instruments she went on to learn from a big variety of musical instruments during her childhood. The result was she grew up to become an accomplished singer with a pro quality style ornamented with Kenturcky oral tradition (Kirby, 2011:pp1-12). Addie went on to be married to Amos Graham and was blessed with three children Kirby, (2011:6). She and her family lived in Breathitt County before finally moving to Cynthiana Harrison County. In Cynthiana she ran a clothing store together with her daughter before finally resting in 1977. The recording of her first album been a long time traveling by the Appal Record Labe was the bed her recognition this is though she had been singing for very many years. Her songs have gone ahead to be recorded by others like Ginny Hawker, Mike Seeger among others

Individual 3 international trade operation Essay

Individual 3 international trade operation - Essay Example Samson. However, when investing in the international market, Mr. Samson should be prepared to deals with challenge of stiff competition from the international firms (Choudhury, 2012). Therefore, this study will provide an insight of various elements that Mr. Swanson should take into consideration when investing in the international markets. What is portfolio diversification? Explain why are foreign investments effective at diversifying a portfolio? Portfolio diversification entails hedging risk by investing in various assets/portfolios. This means that a diversified portfolio will expose an investor to less risk as compared to a single investment. For example, if one investment is performing poorly in the market the other one might be performing well hence, distributing risk unlike in a situation where one has invested in single portfolio. Therefore, investors are strongly urged to invest wisely by diversifying their portfolio (Hagin, 2004). In above connection, foreign investments h ave been reported to be effective at diversifying a portfolio due to the following reasons. There is well advanced level of information technology that enables investors to track their investments and market trends as well as promotion of idea exchange. International market tends eradicate foreign exchange controls and hence making it quit effective to diversify portfolio, higher growth and proper flow of international capital makes it effective to diversify portfolio in the international market. Development of both multinational and global companies had made it easier and effective to diversify a portfolio in the international market. Trade deregulation in the international market has made it effective for portfolio diversification within the international market (Yavas, 2007). What are the main reasons to invest in international markets? Explain. The main reasons to invest in international market include: risk reduction. For example, if one has investments in Japan and the other o ne in the U.S, economic down town in U.S may only affect an investment that is in the U.S but not the one in Japan. This means that investing in the international market prevent one from incurring greater risk. Secondly, international markets provide an investor with greater investment opportunities. For example, international market provides investors with an opportunity to trade stocks with higher value as compared to those offered in the domestic market and hence, providing an investor with higher returns (Gibley, 2012). Thirdly, an international market has higher growth potential as compared to domestic market. This provides an investor with an opportunity to take advantage of potential growth in the foreign markets. Fourthly, International Markets provides an investor with an added advantage because international companies can help to boost returns especially when there is fluctuation of domestic currencies. For example, an international investor may obtain more returns when th ere is a decline in the value of dollar while on the contrary a domestic investor may obtain losses. Therefore, the above benefits can lead an investor to venture in international market (Gibley, 2012). What are the major risks associated with investing internationally? There are three most common risks that may be experienced when investing in the

Cross Culture Communication Essay Example | Topics and Well Written Essays - 2250 words

Cross Culture Communication - Essay Example Emphasis on universal national values of culture suggests that individuals are able to shape their values from experiences gained within a society (Kim, 2008:44). When studying values as noted by Schwartz it comes out that, a bipolar relationship can only exist between and among a number of cultural values. This paper will present a personal reflection on the experience of working in the cross-cultural team and managing the cross-cultural team. During the research when holding the managing post for various sessions, it is worth noting that the task of organization and management in a firm relies heavily on communication between and among all employees present in that firm. The organization should exhibit a great knowledge and application of bipolar relationship whereby conservatism and autonomy dominated the task. Since conservatism and autonomy highlight that the nature of a relationship exists between individuals and a group, it is upon the managing team to ensure communication flo ws effectively. This means that as a manager, one must break down all barriers to effective communication (Fabbi, 2011:79). As such, there are platform that highlight and note presence of effective communication tools. ... Apart from that, from the experience of managing it is worth noting that for employees to perform efficiently, they need motivation. Motivation is a key factor towards realizing the best out of the available employees in an organization (Reisinger, 2009:57). When a manager recognizes efforts of employees and subsequently awards him or her, their output increases automatically. This shows that elements that boost the morale of employees are important as have the capability of taking the organization to the next desired level (Norales, 2006:152). During the management session, a number of issues related to cross culture arise. For instance, since culture is a broad subject, it becomes clear that some employees might not understand particular values and attitudes of the organization (Gross, Jan, and Paula, 2009:67). Furthermore, there are barrier in terms of technology and material culture within and among different employees all in the same organization. Aesthetics and education are al so part of the issues related to cross culture that have chances of arising during the learning sessions (William, 2003:302). Nevertheless, given the point that there are means in which managers can deal with issues arising from cross-cultural dimensions, my learning experience points out that organizational training and practice can be part of the solutions to a large number of cross-cultural issues (Wu, 2008:77). Through performance orientation, it will be easy to deal with the erupting issues. Besides, presence of more orientations based on humane, collectivism, and future prospects makes the procedure of handling any issues arising from cross culture more efficient (Wu, 2008:100). As a result, experience based on management of

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).

Themes and Characters in For Whom the Bell Tolls Essay -- For Whom the

Themes and Characters in For Whom the Bell Tolls For Whom the Bell Tolls, by Ernest Hemingway, is a contemporary novel about the realities of war. The novel is wrought with themes of life and stark direct writing. The characterization in the story is what comprises the intricacy of the underlying themes within the tale. The story itself is not complex, but the relationships of the characters with the environment and with each other coupled with Hemingway's command of description and understanding make the novel as a whole, increasingly developed. The emotions of the story are not found in the dry narrative but rather from the character's themselves. The main character, Robert Jordan, has personality traits spanning various aspects of the heroic side of human nature. In addition, he displays ingenuity and perfectionism. His actions also show a high degree of introspection and philosophical thought. His relationship with Maria and the conflict it causes results in Robert Jordan's discovery of his personal values. He struggles to understand what defines his life and resolve the conflict of what to live or die for. Other secondary characters within the novel are Maria, Pilar, and Pablo. Pilar and Pablo play pivotal roles in both the story and the development of Robert Jordan's character. Their personality traits come into direct conflict with each other, affecting Robert in a wide variety of ways. Pilar can be best described as an aggressive, dedicated, outspoken women who feels comfortable leading a group or controlling a situation. Pilar demonstrates her skill at various times within the text, most notable ... ...xual trauma and made a woman by Robert, and he is given true happiness by her. Indeed, the rarity of their love is apparent when one analyzes the diction and syntax describing their lovemaking: lightly, lovingly, exultingly, innerly happy and unthinking and untired and unworried and only feeling a great delight and he also said my little rabbit, my darling, my sweet, my long lovely. The repetition of word structures and then sentence structures creates a catharsis. The repetition of words beginning in "l" and then "u" establish a parallel sentence structure which creates a rhythm alluding to their own physical interaction. They fall in love, these two people, one always looking ahead and the other always looking back. Through the necessity of war and the help of Pilar, they are able to learn to live in the now, and through this learning are able to grow as characters.