Amazon Swot Analysis

The company’s positioning and business model * Amazon is the first mover of virtual retail supercenter selling products which started off from selling only books online and also offer the lowest possible prices to its customers. * Thus, Amazon is likely to be seen as a ‘broad differentiator’ who is in the middle of differentiate and cost leaders approaches. * However, it can be seen as more leaning to differentiators as most of its past and current strategies are focusing more on innovation of services to its customers.For example, Amazon Fresh for grocery, 1-Click Checkout system, cloud computing and storage etc. * Amazon apply the second approach for market segmentation; it recognize the different customer groups and offers as various products as possible to the customers to attracts more sales and gain more market shares. Successful strategies * First mover of IT retailer shop, popular feature on website such as product reviews, rating * 1-Click system, Mix pur chase, The Amazon Associate program * Growing to sell music CDs, then electronics products and so on. Avoid distribution and stocking cost strategy: Free shipping deal/promotion, cooperate with B&M companies so buyers can pick up product at B&M stores. * Online services for small to medium business, sell its SOA software services * Zshops for used products, and Amazon Web Service These strategies allowed Amazon to obtain its competitive advantage over its rivals, as it’s a unique and utilize the existing distribution channel and IT services advantages Failure strategies Online auction; lost to eBay and the market trend ( fixed-price market booming) * Unbox, Search engine market (track its customer across WWW) * Amazon Prime and Kindle book reader; lost to Google free online data storage and iPad These failure strategies result in declining in stock prices and investing funds, which can be huge in some of the project ie. Kindle, It can be seen as inefficient move which can be avoid if Amazon had done more planning and market research beforehand.Recommendations * Amazon should focus more on cost controlling strategy because the persistent issue for Amazon is high value chain cost, such as IT infrastructure cost, innovation investing, and operating cost. * Amazon can also put more effort on proper market research or planning strategy for shareholder interests, as the CEO seem to be having fun to innovate new services and learning from the market responses, either good or bad, he seem to be okay with both as a learning experience.

HR manager

As the Assistant to the HR manager, I would make a mandatory requirement that all incoming employees must complete a new program that was created by me with in their first 90 days. This program would be designed to ensure that our new talent understands the importance of working together. My program would include these three key principles of working together. The first principle would be on how to effectively communicate as a team. It Is very Important when working together as a team that all vital information Is communicated amongst each other In a clear positive manner.The next principle I would focus on In my program would be having ur Incoming employees work on a group trust exercise. To ensure a high performance team when working together, the team members must be able to trust all the Individuals In the group. The last principle I would Implement Into my program as the assistant to the HR manager would be a diversity workshop. This workshop will prepare Incoming employees to e ngage creatively, and work out difficult situations with peers of many different backgrounds. ncoming employees will find out immediately that there is a major focus on team work throughout their training. In their first week I would have them focus on how to effectively ommunicate to their peers in the workforce. I would set them up in groups, and have them start with ice breakers, then move on to a small collaborative project. I hope that they learn that effective communication is very important, not only to share the proper amount of information, but by also knowing how to talk in a professional manner.You always want to avoid talking down, and barking orders to your co- workers when working together. Effective communication is not possible if you do not talk to your team members in professional manner. Once the new incoming employees have learned how to communicate effectively when working together, I ould then have them participate in trust building exercises. We all know that trust is the building blocks behind any good relationship, and I would want our new incoming employees to have a good working relationship, and to have trust amongst each other.Some of these trust building exercises would include group projects. These projects are to be designed to have each member of the group to complete a certain task. To accomplish the end result, the overall goal everyone will need to play apart. Groups that complete the project will see that their trust and teamwork lead them to their completion. The other groups that did not finish will also see how Important trust and teamwork is to their success. The last key principle I would Implement in my program for new Incoming employees Is a diversity workshop.Diversity In the workplace has many benefits to It! Some key points that I would want my Incoming employees to get from the workshop will be working together with other cultures to foster a more creative and Innovative workforce. Multicultural employees bring a variety of bring new Ideas, and suggestions to the table. Many Ideas that are refreshing, and sometimes very Innovating. I would want the Incoming employees hat are hired for leadership or managerial posltlons to really focus on diversity when Interviewing, and recruiting talent.Recrultlng from a diverse pool of candidates means a more qualified workforce. A diverse and inclusive workforce also helps Duslnesses av010 employee turnover costs I do believe that my program that will be rolling out to our incoming employees will not only be beneficial to their personal professional development, but also to our organizations. The three key principles listed in my program will have our staff prepared to work together as a highly motivated efficient team!

Personal Statement for University of South Alabama

For University of South Alabama - Personal Statement Example The pursuit of this program is also meant to help me develop better management competencies. One of my objectives that will help me develop better management competencies is to utilize the resource that this program will give me in getting a good grip of how to disseminate nursing practice both in writing and orally. My long-term goal is to join the nursing faculty in order to help impart nursing students with specialized knowledge preparing them for quality nursing practice. This is with the recognition that the student enrollment into nursing schools is growing with time whereas the nursing faculty workforce has remained the same. Teaching nursing will enable me to engage nursing knowledge at a higher level, imparting students with skills that will enable them to manage knowledge in this diverse science. Another thing that motivates me endeavor to join the nursing faculty is the increasing complexity of the healthcare system because this is creating a need for nursing students to get specialized knowledge to methodically navigate the system and still maintain high levels of nursing care to patients. This program offers a specialty in clinical nurse specialist in adult health and as such, it is best suited to help me realize my educational goal of gaining better knowledge in adult care. The program will impart me with competence in designing, implementing, managing and evaluating projects aimed at giving better care to adult patients guided by evidence-based practice. Some adult care initiatives needed inter-professional teams and the curriculum of this program is designed in such a way that will give me knowledge on how to lead and manage such teams. While on my clinical rotations, I realized that information technology has become crucial to clinical decision-making that helps improve the quality of patient satisfaction and this is the knowledge with which this program

The Articles of Confederation Versus the Constitution Assignment - 9

The Articles of Confederation Versus the Constitution - Assignment Example In order to understand the differences between these two historical and pivotal documents is to discuss them more deeply and individually. The Articles of Confederation were enacted in March of 1781 and were maintained until 1789, nearly 8 years to the day (Kelly). The Articles were chosen as ideal for a fledgling and inexperienced government to have a better chance of winning the Revolutionary War and defeating the British forces. However, many of the colony’s settlers had a negative experience with governments who had too much power and control and no real experience in the forming of government (Morgan 1). They specifically wanted a government with limited power. Many commented that the Articles of Confederation were essentially a â€Å"friendship agreement† between the 13 original colonies in the war against a shared enemy (Painter 3-4). The Constitution replaced the Articles in 1789. The Constitution was considered to be a â€Å"progressive† document; one th at represented a long term government that was different than those the settlers left behind. This is one of the first things that separate the Articles and the Constitution. The Articles intended to facilitate a favorable end to the war and the latter was intended to develop a long term, stable government structure (Winkler 1). However, the differences go much deeper.  There are several specific differences between the two documents. First, in the Articles, the individual states are sovereign and not accountable to the federal government, whereas the Constitution shares the powers between states and the federal government.

Cellular Network Comparisons Essay Example | Topics and Well Written Essays - 1000 words

Cellular Network Comparisons - Essay Example Some the cellular networks include; UMTS, EV-DO, LTE, WiMax and Satellite (Kostas 38)2. The UMTS (Universal Mobile Telecommunications System) is known as the third world (3G) wireless frequency that is used on the (GSM) Global System for Mobile communication, to convey text, audiovisual, digitized voice as well as multimedia (Markus 62)3. The network architecture of UMTS entails simply of enodeBs continuously on the network. The functional components of UMTS are User Equipment (EU), the essential network as well as the Radio Network System (RNS) which delivers then bring about air interface fort and the call processing of UMTS involves controlling the call operating layer nodes. The weakness of UMTS might be distortion and weak signal while its strength is linked to the high speed to transfer voice as well as mobile data (Markus 93).The EV-DO (Evolution Data Only or Evolution Data Optimized) is a procedure intended to transfer high speed moveable data as well as voice (Markus 72). EV -DO network architecture is centered on wireless Internet Protocol architecture while the functional components of EV-DO are the Core Network (CN), the Radio Access Networks (RAN) and the Mobile Station (MS). The call processing are boosted by IS -95 over high speed data period. The weakness of EV-DO may be distortion as well as weak signal while its strength is allied to the high speed to convey voice plus wireless data (Markus 493). LTE (Long Term Evolution) is a  standard wireless communication, legally submitted as a contender  for a 4G  structure to  ITU-T. LTE is still on Continual architecture network improvement radio technology and its functional component was tested publicized and the achievement of a 4G trial where it succeeded an extreme  packet  its strength broadcast rate of about 5  Gbit/s in the downlink consuming 100  MHz frequency bandwidth to a wireless place stirring at 10  km/h while LTE weakness might be distortion as well as streaming signal. The WiMax refers to Worldwide Interoperability for Microwave Access, the WiMax network architecture is built on the Internet Protocol based where the network is split into three rations called ASN (the access service networks,) the mobile stations (MS) and the connectivity service network (CSN) which runs Internet Protocol linkage (Goff 77)4. The functional components of WiMax cellular network are the BS (base station), the connectivity service network (CSN) as well as the ASN-GW (access service network gateway) (Goff 66). Call process of WiMax session is started once the mobile station (MS) effectively complete the method of linking into the network, thus necessitates opening BCE Basic Capability Exchange, the recording and verification and Internet Protocol obligations. WiMax Strengths is that it is easily accessed, inexpensive and applied effortlessly, WiMax has a wireless broadband that alternate to Cable (Goff 300) while WiMax weakness is that the set up cost is costly, strang ely limited personnel may be capable, and WiMax networking safety might be weak (Goff 340). Satellite is a technology used to communication and is positioned into orbit by human endeavor. The functional component of human made satellite in the delivering of its mandate to cellular networks is to offer communication proficiencies

Enterprise in action Essay Example | Topics and Well Written Essays - 250 words

Enterprise in action - Essay Example Effectual logic takes advantage of unexpected events in the market. On the other hand, causal logic entrepreneurs engage actively in exploring existing opportunities and are capable of building their future upon them. These investors are able to correlate between one variable and the other and interpret the future depending on the consequence that one variable has over the other. When it comes to basis for taking action, effectual logic investors do not make goals at the beginning but the goals tend to emerge in the course of action. In contrast, causal logic is goal oriented, their means of achievement is determined by a set of goals, and objectives that they strive to satisfy. Causal logic entrepreneurs are driven into investment by the fact of maximizing on risk adjusted to the returns. Effectual logic entrepreneurs will limit their investment to a certain level of their acceptance in order to give them a chance to participate again next time. Effectual logic is more flexible but less secure while causal logic is not flexible but secure to some extent (Stuart Reed,

A New Tunnel under the Thames Essay Example | Topics and Well Written Essays - 1500 words

A New Tunnel under the Thames - Essay Example 7. Include a set of drawings of your proposed tunnel, including a. a detailed cross section, b. a long section c. a map showing your suggested new road layout and d. a map indicating remedial works to minimise the impact of the extra traffic on the surrounding areas. 8. The site designated for the northern access (between Galleons Reach DLR and the river) is currently a brown field site with possible contamination, which includes an infestation of Japanese knotweed and some fly-tipping which may contain asbestos. Explain the procedures needed to bring the site into a condition suitable for construction. A new Tunnel under the Thames Introduction A tunnel is a subversive vessel, entirely covered a part from openings for entrance and exit, usually from both ends. A tunnel can be constructed for foot passage, rail, vehicle traffic, or a canal. The new Thames river crossing will allow closing of the Wool Wish Ferry. The bridge will relieve congestion for people living adjacent to Blackwa ll. This marks the location of Thames Gateway Road Bridge that was scrapped by the mayor in 2008, following the rejection of the plan by the Inspector at a Planning Inquiry. The tunnel, which will run from Greenwich Peninsula to the royal Docks, is expected to have a capacity of up to 2400 cars per hour in each route. While the link is very much worthy and will perhaps help reduce the severe overcrowding in the city, it will tamper with Blackwall Tunnel, making the suggestion for a Gallions Rach ferry a laughable alternative for the Thames Gateway Bridge. In addition, it raises questions with regards to the future of the current Woolwich Ferry. Supporters of environmentalists are likely to criticize the scheme of the tunnel, alleging that it would bring problems to people of London by creating more traffic, noise and more pollution. Furthermore, if the leaders in this region want to make London one of the world’s greenest capitals, their policies should be much focused (Irsha d, Wagner & Mussger, 2003). Since the era when Brunel made the first Thames tunnel, which was called Rotherhithe Tunnel, the tunnel construction has undergone great breakthrough, especially between 1825 and 1842. Tunnel constructions during these times were done through a rectangular safeguard with a miner revealing the face. The dimensions of the shield were 29ft high and 33ft wide, with brick coating after the miners and an arch brick being constructed. Nonetheless, tunneling will remain a risky activity since the conditions can never be certain, regardless of the number of boreholes that are taken. Furthermore, it is not uncommon to find tunneling gaining more prominence in the next few decades. The proposed Thames Tunnel will occupy a distance of 25 kilometers from west London. It will be 67 meters below the ground and will widely follow the direction of Thames River. According to the environment agency, this tunnel will link 34 of the most contaminated sewer outflows (Munfah 20 03). CDM requirements CDM regulations background Areas where CDM is applicable and those areas where it is not applicable General health and safety practices and laws Recognition of accident prevention, causation and prevention. Risk management practices Control procedures and reviewing and monitoring requirements Roles of different professional Land/engineering survey Land surveyors establish and give recommendations on the

Coronary Artery Disease Research Paper Example | Topics and Well Written Essays - 250 words

Coronary Artery Disease - Research Paper Example It is established that LDL or Low-density lipoproteins are chiefly atherogenic in nature. On the other hand high-density lipoproteins or HDLs are defensive lipids as they aid in LDL metabolism. On oxidation LDLs become tougher to metabolize and become cytotoxic leading to endothelial injury (Pollard, 2009). Chronic Endothelial injury Hypothesis- Endothelium protection and its repair is performed by glycosaminoglycan or GAGs, when GAGs become weak, atherosclerosis is instigated. The process is promoted due to exposure of endothelial cells and free-radical damage. As damage of endothelial lining is initiated the site promotes the permeability of plasma constituents especially the lipoproteins. Thus oxidized LDLs are capable of damaging the endothelial lining of the cells which results in the formation of plaque (Stephen, 2011). When the endothelial cells are injured, the macrophages present in the blood move around from the blood circulation. The inner side of the artery forms a layer called layer of intima. Consequently, smooth muscles join intima along with connective tissue as well as lipids present inside as well as outside the cells to generate a plaque. This brings about union as well as accumulation of platelets, as well as release various growth regulating factors to amplify the procedure ensuing obstruction of the lumen of the artery, leading to thrombus establishment (Mottillo, 2010). Mottillo, S., Filion, K. B., Genest, J., Joseph, L., Pilote, L., Poirier, P., Rinfret, S., Schiffrin, E. L., Eisenberg, M. J. (2010). The Metabolic Syndrome and Cardiovascular Risk: A Systematic Review and Meta-Analysis. J. Am. Coll. Cardiol, 56(14), 1113- 1132. Stephen D. Wiviott, Marcus D. Flather, Michelle L. ODonoghue, Shinya Goto, Desmond J. Fitzgerald, Fernando Cura, Philip Aylward, Victor Guetta, Dariusz Dudek, Charles F. Contant, Dominick J. Angiolillo, Deepak L. Bhatt.

Based on your ideas on Nilsen and Tannen, discuss how language Essay

Based on your ideas on Nilsen and Tannen, discuss how language reflects patriarchy (Patriarchy) - Essay Example Besides, she manages to show that even when filling forms, women tell stories about themselves because most forms offer four titles to choose from and they include â€Å"Mr.†, â€Å"Mrs† or â€Å"Miss† and â€Å"Dr†. In her work, â€Å"Mr.† â€Å"carries no meaning other than that the respondent is male† (Tennen395); however, when women accept â€Å"Mrs.† or â€Å"Miss† it shows that they have been married and that they have conservative forms of address. Women using the label â€Å"Ms.† decline to reveal if they are married while men using the title â€Å"Mr.† decline nothing considering that nothing was asked; hence, women using â€Å"Ms.† tend to be seen â€Å"as either liberated or rebellious depending on the observer’s attitudes† (Tennen395). Tennen shows that married women surnames are marked and that if a married woman accepts her husband’s name, she acknowledges to the world that , â€Å"she is married and has traditional values† (Tennen396). ... From her dictionary cards, Nielsen shows that â€Å"feminine words have acquired sexual connotations while the masculine word retains serious business like aura† (Nielsen). For instance, she manages to show that while a callboy is an individual who calls actors when time comes for them to go to stage, a call girl is considered a prostitute. In her study, Nielsen found about 200 pairs of words that accepted both masculine and feminine forms and in nearly all of the pairs, masculine form was considered the base where a feminine suffix was added. The masculine form is basis on which compounds are made apart from one semantic area where masculine word is not the basis and the areas deals with sex, marriage and motherhood. Nielsen shows that when someone refers to a virgin, the listener probably considers a female unless the speaker specifies a masculine pronoun, which is the same case with the term prostitute. Moreover, Nielsen shows that the grammar used in writing or even talkin g about weddings show the expectation of men playing an active role when men wed women while the women become brides of men. Nevertheless, titles offered to royalty indicate that males can be at loss because of the assumption that they predominantly play a powerful role; for instance, when a female has the royal title, then the man she marries does not automatically acquire a matching title. From Nielsen’s article, it is clear that connotations between encouraging male and pessimistic female connotations can be established from various pairs of words that differ denotatively in the area of sex. For instance, patron and matron have varying degrees of esteem that womenfolk attempt to use the

Statistical Process Control (SPC) Essay Example | Topics and Well Written Essays - 500 words

Statistical Process Control (SPC) - Essay Example In contrast to this, SPC uses statistical techniques to ascertain whether there are any deviations in the production line that may end with the project being rejected. The concept behind the viability of the SPC is that any product in a production process will have a certain degree of deviation in terms of its properties, which will vary slightly from its design values. SPC manages these deviations and prevents them from getting out of control, by analyzing the variances within the process from time to time. For example, by using statistical tools, any person who oversees the functioning of the production line can use them to detect any profound and undesirable changes (which exceed the specified limits). The causes of any such variation could be due to a host of factors such as wear and tear due to continued use etc. and as such the technique offers a great chance of correcting the problem at the source itself thereby ensuring that subsequent products from the production line stay well within the desired limits. In worst cases, this could alert the supervisor so that he/she may stop the production in order for the problem to be rectified.

Individualism and Communilism Essay Example for Free

Individualism and Communilism Essay How far can a person go in life without the need of others? Is it possible for him or her to achieve success without having any sort of social dynamic to aid in the progress of their lives? Does having strong social connections aid a person in shaping his or her character to achieve their goals and can these achievement be defined as his or her own if there was intervention of any sort from a social group? It is possible to deduce answers to such notions upon a better understanding of the distinction of community and the individual and the effects of these ideas on each other. The following essay will try to define the distinction between the ideas of communalism and individualism. It will try to isolate the characteristics of each ideal in order to better comprehend their influence on a person life and try to provide an argument on why these two ideas may need to work together in order for a person to lead a fulfilling life. Communitarianism defines the idea where the individual needs of a person are secondary to the overall development of the social group he or she communes. The social group can be represents as any group of people that share common goals and ideals. Possible factors that define ideals of the group include ethnic background, social or economic status, religious and cultural beliefs. Constant social interaction is important with this ideal. It is communitarian teleology that humankind is social by nature and that this idea grows naturally to this end. Aristotle was a proponent of communitarianism and – according to class lectures on the subject – believed that being part of a community eased the burden of everyday living. He provided an example on the natural development of speech as a direct result of the necessity of humans to interact with each other to participate in a community. He also hypothesized that in order for human development to flourish there needs to be a sharing of skills and ideas within the social group. The contrasting idea to communitarianism is individualism. Individualism focuses on the idea of the promotion of an individual thoughts and desires before those of a social group. This idea believes that external involvement from others is unnecessary when trying to achieve an individual’s goals, unless the external interference aids in the achievement of the individuals goals. Advocating social isolationism is not a necessary factor under this ideal; rather the individualist is often part of a social group and adheres to the communal laws and practices set forth upon them in order to promote their own personal gain. Thomas Hobbes defines an extreme extend of complete individualism. He states, that under individualism, humans care only about themselves and have no regards for the necessity of others and have no moral concern for their actions. Under this state of reality, it seems highly unlike for human development to expand as all focus lie on achieving individual goals for the sake of individual gain rather than sharing the results of intellectual, social, economic, etc. endeavors. Hobbes realized that such a state of reality cannot naturally exist and deduced that the individualist enters society and agrees to abide by its rules and regulations with the sole purpose of expanding their own personal goals; he coined this compromise â€Å"the social contract. † Based on the interpretations of the ideals of communitarianism and individualism it is possible to obtain an idea on how these ideals shape the individual’s outlook on life. Communitarianism appears to promote the well-being of all, the question to this is at what cost? The idea that the needs of the many are more important than the needs of the one fails to account for moral responsibility. If we explore individualism further and do not use the extreme representation of the term, we realize that being an individual actually means being free to make choices outside of what other people expect or dictate. In other words, by being an individual you are free to choose and are directly responsible for your actions and their moral implications. Being an individualist is to have free will. It is impossible to deny that humans tend to gravitate toward the state of a social-collective and in fact, there is evidence that social interaction is necessary. For example, as children we tend to obtain knowledge from many others. We do not learn from one person or one experience, rather we continue to expand our knowledge and thereby our self. In this sense, communitarianism works by providing the individualist with the tools to make choices and commit to actions, but it does not dictate how this person will use the knowledge. It is up to the individual and his singular choice to decide how to use this knowledge. Communitarianism may hinder personal development. If the individual considers the needs of his community, before his own how can he or she expect to develop as a person? In fact, in some cases, the community will try to keep an individual from growing. For example, in certain communities poverty is the prevalent norm. If the person acts to try to remove him or herself from such a community through his own achievements, some in such community may see him in a different light, not wanting to be part of the group anymore. This sense of guilt can weight on the individual and create doubt on his achievements and on himself. Although he or she was free to make the conscious choice to get out from poverty, the community still has an indirect control over the person. Communitarianism may also interfere with a clear sense of moral judgment. In general, a person’s self-worth and values are defined by their individual actions. In some cases, communitarianism blurs the distinction between the values of the individuals and the values of the group. In such instances, wrongful generalizations of a person values and choices can be made based solely on the choices and values of the community they belong to; this is called stereotyping and/or racism. It is unclear how the will of others can influence the moral decisions of a person. Communitarianism can server a person in the early stages of life (when individual thought and reasoning are not fully developed) to provide the tools necessary to shape their life. However, in the end, the individualistic actions of a rational, independent, free-to-choose individual that define a person.

Isolation of Recombinant Escherichia Essay Example for Free

Isolation of Recombinant Escherichia Essay One technique important in both genetics and biochemistry is the Polymerase Chain Reaction (PCR), first developed in the 1960s, and then automated in 1983. Current PCR technology was not developed until the discovery of thermostable polymerases, specifically Thermus aquaticus (Taq) polymerase (1). The protein Taq polymerase was first isolated from the extreme thermophile T. aqauticus, where extreme thermopiles are bacteria that live in temperatures at or above 45 °C. The Taq enzyme is a member of the DNA polymerase I family (2, 3). The interesting property of Taq polymerase is that it has a temperature optimum at 74-75 °C, allowing it the remain active in temperatures required for PCR double stranded DNA denaturation (3, 1) . The protein has an approximate molecular weight of 6263 kDa when isolated from T. aquaticus, and 94 kDa when isolated from recombinant Escherichia coli, and is still stable at temperatures of 93-95 °C, hence the thermostability of the enzyme ). Taq specifically lacks any proofreading activity in the 3’ to 5’ direction, and therefore has a relatively high error rate of single base mispairings of 1 error per Isolation of Recombinant Taq Polymerase for PCR 9000 nucleotides, as well as a frame shift error rate of 1 per 41,000 basepairs (5, 6). Taq polymerase has an activity that is highly dependent on the environment of which it is in as it is thermostable, and has differing activities at nearly all temperatures up to the point of denaturation. Taq specifically can add up to 1000 base pairs in length on a template in under one minute under typical PCR conditions. The enzyme has a specific activity of 200,000 units mg-1, and can add approximately form 60 nucleotides per second at 70 °C (7). The isolation of Taq is essential for the PCR reaction. The most important reason for Taq being used in PCR is the thermostability at high temperatures (95 °C). This allowed for the process of elongation, annealing, and denaturation to occur without the replacement of new enzyme, and thereby, was more efficient, faster, and cheaper because the reaction could be automated through the use of a machine known as a thermocycler which basically is just a machine able to change temperatures of an isolated environment rapidly (7). Prior to the discovery of Taq, PCR was done using Klenow fragments of E. coli DNA polymerase I at 37 °C. The lack of thermostability required replenishment of enzyme after each PCR cycle (8). One of the initial difficulties of Taq polymerase was the organism in which it was expressed in, T. aquaticus, as it was difficult to culture and produce large quantities of enzyme. E. coli bacteria were engineered to expressed the Taq polymerase gene to allow for retrieval of large quantities of enzyme ). The isolation of the Taq gene involved culturing T. aquaticus and then isolated the DNA of the cells through lysing, proteinase K addition, extracting of aqueous and phenolic phases, dialyzing of extractions, addition of SDS, and then centrifugation of solution to eventually retreieve the DNA of the organism as outlined in Lawyer et al., 1989. With the isolation of the 2401+ BP gene of Taq, the gene was incorporated into a 6.58 kbp plasmid (pLSG1). The gene was inserted 171 bp distal to the lacZÃŽ ± promoter/operator, and 109 bp distal to the BgII site, so the gene expression could be controlled through an inducible promoter. With the pLSG1 plasmid, the vector was introduced to E. coli bacteria to allow for plasmid uptake (4). Other experiments have been conducted towards the purification of Taq from recombinant E. coli. Specifically Engelke et al., 1990 developed a method for purfication of Taq. The E.coli strain 2 DH1 was used for the expression of the recombinant plasmid containing Taq polymerase. The bacteria were grown in 12 Litre batches of Luria Broth; using 1 mL of saturated DH1 culture and 80ÃŽ ¼g/mL of ampicillin. Isopropyl-1-thio-ÃŽ ²-Dgalactopyranoside (IPTG) was added to 0.5mM and the cultures were grown for 16-20 hours. The cells were harvested in 2.4 L of buffer A (50 mM TrisHCL, pH 7.9, 50 mM dextrose, 1mM EDTA) and collected via centrifugation, resuspended in Buffer A with 4mg/mL lysozyme and incubated at room temperature for 15 minutes. Buffer B (10 mM TrisHCl, pH 7.9, 50mM KCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Tween 20, 0.5% NP-40) was added and incubated in 180 mL fractions, for 60 minutes at 75 °C in a water bath. The mixtures were centrifuged at 8000 rpm for 15 minutes at 4 °C. Taq then precipitated with polyethyleneimine (PEI) at room temperature, then isolated through centrifugation and suspended in buffer C (20mM HEPES, pH 7.9, 1 mM EDTA, 0.5mM PMSF, 0.5% Tween 20, 0.5% NP-40) containing 0.25 M KCL. PEI eluatents were diluted in 50mM KCL and buffer C and applied to a 150mL BioRex 70 ion exchanger column, and then eluated using 200mM KCL. The protein was dialyzed for 12 hours against two changes of 1 L storage buffer (20mM HEPES, pH 7.9, 100 mM KCL, 0.1 mM EDTA, 0.5 mM PMSF, 1mM dithiothreitol, 50% glycerol. The experiment resulted in 40-50 mg of protein per litre of cell culture (9). The methods used in this experiment differed in certain key aspects. First, Engelke’s experiment made use of a higher concentration of ampicillin. The IPTG was added to the same concentration, but was added after cell growth up to an optical density of 0.700. Instead of a water bath at 75 °C, this experiment made use of an air incubator for the temperature requirements. Engelke’s experiment made use of PEI to precipitate Taq, while this experiment made use of 30g of (NH4)2SO4 per 100mL of supernatant. Buffer C was not used throughout this experiment, and no ion exchange columns were used. The dialysis procedure was done for twice as long with twice as many changes of solution per 6 hours. The changes made from Engelke’s experiment offers a different method for protein precipitation. The method used by Engelke made use of PEI which is an affinity precipitation method versus a salt prec ipitation method. The PEI Isolation of Recombinant Taq Polymerase for PCR method has the major drawback through the lack of selectivity, and can often precipitate nucleic acids as well (10). This is why the BioRex column needed to be used. Ammonium sulfate has the advantage that the precipitation can be controlled based on ionic strength of species involved, as well as has no negative effects on the activity of the target enzyme. Salting out also has the advantage that only native state proteins are precipitated due to the hydrophobicity involved with native state proteins (10). Buffer C was not required for this experiment as no BioRex column was required. This experiment made use of various techniques and methods including: SDS-PAGE, differential centrifugation, Western Blotting, real time-PCR (rtPCR), PCR, agarose gel electrophoresis, and dialysis. Two important techniques were PCR and rt-PCR. PCR does not allow for the quantification of DNA amplicons as it is an end-point PCR, but it does allow for confi rmation of template duplication along with measurement of base pair length. Amplification of primer would confirm the presence of a thermostable DNA polymerase. The following agarose electrophoresis helps to find amplicon size which can tell us the activity of Taq, as well as the specificity, as one template should only return one band in PCR (7). rt-PCR allows for a quantitative assessment of PCR, and therefore the kinetics of the reaction, as it detects the amount of amplicons produced in the reaction. The point at which the standard curve reaches threshold in cycle number gives information on the activity of Taq, as a more active sample of Taq reaches threshold earlier. Melt curve analysis also provides information regards DNA amplicons in solution (11). The purpose of this experiment was the test the methods for the isolation of PCR grade Taq polymerase from recombinant E. coli using differential centrifugation, salting out, and heat denaturation following lysation of cells to potentially improve isolation of Taq from past methods. The presence of Taq will be confirmed through Western blotting, and rt-PCR and PCR reactions along with purity will be assessed through SDS-PAGE. The activity of Taq will be found through rt-PCR and PCR. Finding the most efficient method for the isolation of Taq offers a valuable reagent source for any PCR reactions required. The isolation technique would also be applicable to any thermostable proteins. 3 EXPERIMENTAL PROECDURES Isolation of Taq Polymerase Luria broth (500 mL + 100ÃŽ ¼g/mL ampicillin) was inoculated with 50 ÃŽ ¼L of frozen Taq polymerase expressing E. coli cell stock. Incubation was commenced for 12 hours at 37 °C until the Optical Density had reached 0.700. IPTG (0.5 mM or 0.112g/L culture) was added and the culture was incubated for 12 to 14 hours at 37 °C. The 50mL of cells were then centrifuged (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810, and 5 mL of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1mM EDTA) was used to suspend the separated pellet. The solution was then centrifuged again (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810 and the pellet was once again suspended in Buffer A, with an additional 20 mg of lysozyme added. The reaction was incubated for 15 minutes at room temperature. Following incubation, 5mL of buffer B (10 mM Tris HCl, pH 7.9, 50mM KCl, 0.5% Tween 20, 0.5% NP-40, 1mM PMSF, 1mM EDTA) was added and incubated at 75 °C for 1 hour in a New Brunswick Scientific-Innova 40 incubator shaker series, and shaken by hand approximately every 5 minutes. The solution was then centrifuged (15000 RPM x 10 minutes at 4 °C) in a Thermoscientific Sorvall RC 6+ centrifuge and using a 603s Delta Range 30g of (NH4)2SO4 per 100mL of supernatant (8 mL of supernatant equivalent to 2.4g (NH4)2SO4 ) was added and incubated for 10 minutes at room temperature and shaken on the Innova 40 incubator. The lysate was then centrifuged again (15000 RPM x 10 minutes at 4 °C) in Thermoscientific Sorvall RC 6+ centrifuge and the resultant pellet was suspended in 2mL of buffer A. The solution was then dialyzed in a Spectra/Por membrane tubing set at 6000-8000 Da molecular weight selection in 1 L of storage Buffer (50 mM Tris HCl, pH 7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5 mM PMSF, 50% glycerol) for 24 hours at 4 °C changing the buffer every three hours. The dialysis solution was then diluted in a 1:1 ratio of storage buffer and stored at -70 °C until needed. Protein Concentration Determination A Bovine Serum Albumin Bio-Rad assay standard curve was prepared (0 –0.3 mg/mL) using Isolation of Recombinant Taq Polymerase for PCR a 1mg/mL stock solution and an Asys Expert Plus spectrophotometer set at 620 nm. Bio-Rad assay was run in triplicate using 20ÃŽ ¼L of protein dilution and 150 ÃŽ ¼L of diluted Bio-Rad Dye Concentrate. 10x and 100x dilutions of the sample prepared previously were made and 20ÃŽ ¼L were used with 150ÃŽ ¼L of diluted Bio-Rad Dye concentrate. The solutions were incubated for 10 minutes and absorbances were tabulated. sandwich was then assembled with an additional ice block in the transfer apparatus. The apparatus was run at 180mA overnight in a refrigerator and the membrane was then stored in TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and refrigerated. 24 hours prior to the primary antibody (Anti-Taq monoclonal Antibody (8C1)) addition, the membrane was blocked in 1 gram of Carnation nonfat dry milk (5% w/v) and 20 mL of TBST Buffer. The primary antibody in TBST with SDS-PAGE 5% w/v nonfat dry milk at a 1:800 dilution of A discontinuous polyacrylamide gel was antibody was added to the membrane and shaken prepared using a Mini-PROTEAN Tetra Cell for 1 hour at room temperature. The membrane was module. The casting stand was assembled for 1mm then washed three successive times for 15 minutes gel and filled with National Diagnostics 12% with TBST buffer at room temperature. The Resolving Gel (Protogel 2400 ÃŽ ¼L, Resolving Gel secondary antibody (Peroxidase-conjugated Buffer pH 8.8 1560 ÃŽ ¼L, dH2O 1974 ÃŽ ¼L, 30% w/v AffiniPure Goat Anti-Mouse IgG (H+L)) was then APS 21ÃŽ ¼L, TEMED 6ÃŽ ¼L), casted to 1 cm below top applied in TBST with 5% w/v nonfat dry milk at a of glass plate, and then 4% Stacking Gel (Protogel 1:2000 dilution of antibody and shaken for one hour 390 ÃŽ ¼L, Stacking Gel Buffer pH 6.8 720 ÃŽ ¼L, dH2O at room temperature. The membrane was then 1830 ÃŽ ¼L, 30% w/v APS 6ÃŽ ¼L, TEMED 3ÃŽ ¼L) was washed three successive times for 15 minutes with casted on top with a ten well gel comb. The TBST buffer at room temperature. The edges of the electrode set up was then submersed in 1 x Trismembrane were dried with a Kim and next 1mL of Glycine SDS Page Running Buffer. To each 20 ÃŽ ¼L Renaissance Western Blot Kit NEN Life Sciences of sample, 20 ÃŽ ¼L of Laemmli buffer (0.5 M TrisProducts, Cat. No. NEL 101 luminol reagent with HCl, pH 6.8, 4.4% w/v SDS, 20% v/v glycerol, 2% 1mL of oxidizing reagent were mixed together and v/v 2-mercaptoethanol, 10 mg/mL bromophneol then applied to the membrane. The membrane was blue) was added and boiled for 3 minutes and then then imaged with an exposure time of 40 seconds cooled for 5 minutes on ice. To one well 7ÃŽ ¼L of using AlphaEaseFC software. New England BioLabs Inc. Prestained Protein Marker (7-175 kDa) was added. To the following PCR/agarose gel electrophoresis wells 15 ÃŽ ¼L of standard Taq polymerase was added, A master mix for PCR was prepared (1x PCR along with 20ÃŽ ¼L of six different samples, with the buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, fifth being prepared the previous year with the same 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 method of isolation as outlined previously. The gel ng Template DNA and Nuclease-free PCR water) was run at 200 Volts for 40 minutes, incubated in and 22.5 ÃŽ ¼L of master mix and 2.5ÃŽ ¼L of Taq fixing solution overnight and then stained with Bio- sample, the standard, or the Taq prepared a previous Safe Coomassie Blue for one hour at room year were added to PCR tubes and centrifuged temperature under agitation. The gel was then briefly on a Fisher Scientific Accuspin micro 17 analyzed used AlphaEaseFC software. just briefly using 1.5mL eppendorf tubes with no caps to contain the PCR tube. The PCR tube was Western Blotting then added to T3 Biometra Thermocycler and Using the method described above for SDSdenatured at 94 °C for 3 minutes and then 35 cycles PAGE, a SDS-PAGE gel was taken prior to fixing. of PCR with the denature 94 °C for 45 seconds, The gel was then transferred to transfer buffer anneal 55 °C for 30 seconds, and extension at 72 °C (20mM Tris-HCL, pH 8.0, 150 mM Glycine, 20% for 1.5 minutes. The sample was then incubated at Methanol). Immobilon-P transfer membrane with 72 °C for 10 minutes and then temperature was 0.45 ÃŽ ¼m pore size and Whatman paper were cut to maintained at 4 °C. The samples were then stored at the size of the gel. The membrane was wet with -20 °C until agarose gel preparation. A 1% agarose 100% methanol, then transferred to MilliQwater gel w as prepared through 1.5g of agarose (Sigma and soaked for several minutes. A standard blotting No. A-6877 Type II) to 150mL of Tris-Acetate4 Isolation of Recombinant Taq Polymerase for PCR EDTA (TAE) buffer. The solution was microwave for 1 minute and mixed until in solution. Once cooled to 60 °C, 7.5 ÃŽ ¼L of Biotium Gel Red Nucleic acid stain was added and mixed. The solution was then poured into the electrophoresis tray; a comb was installed, and set at room temperature. One Litre of 1x Tae buffer was prepared through dilution of 50x TAE buffer and then the solution was poured onto the electrophoresis tray to cover the gel in 1mm of buffer. 20 ÃŽ ¼L of PCR product prepared previously and 4ÃŽ ¼L of Gel Red dye were mixed and 20ÃŽ ¼L of each sample, the standard, and Taq prepared the previous year and Invitrogen life Technologies 1 Kb DNA ladder Cat. No. 15615016 was run at 150 Volts, 100 mA for one hour (or until dye reached the bottom of the gel). The bands were then visualized under 300 nm light and fluorescence was measured at 590 nm. The gel was analyzed using AlphaEaseFC software. concentration of the sample Taq was 1.88 + 0.11 mg/mL. The solution of proteins was not pure Taq as confirmed by the SDS-PAGE (Fig. 2) as various proteins created distinct bands (B to K excluding E). The standard Taq revealed only one band (A), indicating band E was most likely belong to Taq, as it was the darkest band in the gel. An analysis of the molecular weights of the bands through electrophoretic mobility (Tab. 3) showed the standard Taq having a molecular weight of 115.2 + 14.6 kDa, and the likely band (E) had a molecular weight of 113.4 + 14.3 kDa. There was a distinct distortion in the bands of the SDS page in all lanes with the exception of the standard Taq and the 2011 Taq (Fig. 3). The distortion is of a smile. The overall gel also has a large distortion, but of a frown. It would appear there was a similar protein to D E and F present in all samples, including the 2011 sample. The standard Taq did not contain the bands. Re al Time PCR The Western Blot (Fig. 4) revealed distinct A master mix for PCR was prepared (1x PCR bands; however, there were more than one band in buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, each lane with the exception of the standard Taq. 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 Two distinct bands were present in 5, Taq, and 2 (b, ng Template DNA and Nuclease-free PCR water). c). The lanes of * and ? contained several bands To PCR tubes, 22.5 ÃŽ ¼L of Master Mix and 2.5 ÃŽ ¼L also. The overall gel also expressed a slight color of Taq sample or the standard Taq were combined, banding along the solvent front edge which is mixed through vortexing and then centrifuged with shown in both Fig. 3 and 4. The 2011 lane did not a Fisher Scientific Accuspin micro 17 just briefly appear to have any Taq present, as no band was using 1.5mL eppendorf tubes with no caps to distinguished. The entire ladder expressed some contain the PCR tube. The Taq samples were antibody activity. prepared in triplicates. 20ÃŽ ¼L of each sampled were The real time-PCR revealed a threshold reached then transferred to a 96-well PCR plate and then at 20 cycles, with the vast majority occurring at 24 sealed. The well was then placed in a BioRad CFX cycles. The melt curve showed an approximate connect Real Time System using the programing of melting temperature of 81 °C (Fig. 7). enzyme activat ion (95 °C, 30 seconds, 1 cycle), 40 The agarose gel electrophoresis revealed one cycles of Denaturation (95 °C, 1 second) and distinct band at approximately 5883.5 base pairs in annealing/extension (60 °C, 5 seconds), with a melt length. The brightest bands, and therefore the curve of (60-95 °C in 0.5 °C intervals, 3 seconds per highest quantities of Taq enzyme were found in the step, 1 cycle). The samples were then analyzed std., 2 and 4. When the base pairs specific activity using AlphaEase FC software. of the enzyme was calculated it was found to be 834.5 + 63.9 bp/min/ÃŽ ¼g of sample, or 3922.3 + 192.9 bp per minute. RESULTS The results of the Bio-Rad assay on the sample of Taq polymerases diluted to 10x and 100x revealed that the 10x dilution was far to concentrated and fell outside the linear curve of the Bio-Rad assay. The retrieval of protein from the Luria broth was found to be 300.8 + 17.7 mg protein per L of Luria broth. These results (Tab. 1) suggest the protein 5 DISCUSSION Through the analysis made through SDS-PAGE, the MW of the standard Taq was found to be 115.2 + 14.6 kDa and 113.4 + 14.3 kDa. This is different from the accepted literature value of 94 kDa (9). Even with error correction, the prot ein did not fall Isolation of Recombinant Taq Polymerase for PCR within the range of the accepted literature value. In total, the two proteins differ by 23% and 21% without error correction, or 21.2 kDa and 19.4 kDa respectively. In comparison to one another, the two bands have essentially the same molecular weight, indicating whatever error occured in the gel was equivalent on both the standard and the isolated Taq. One explanation for the difference in the molecular weights may be explained through the quantitiy of protein used. The darkest and thickest band ( E, fig. 4) likely belongs to the Taq protein. To get a more defined band, a dilution would be effective in making a higher resolution band (12). The amount of protein isolated per volume of Luria broth was determined to be 300.8 + 17.7 mg per L of Luria Broth. Quite obviosuly, there are issues both with the heating of the gel, and distortion of the bands into â€Å"smiles†. The distoration of the gel likely was caused by unequal heati ng of the gel causing the center of the gel to be hotter than the peripheries, as the walls of the apparatus act as heat sinks (13). The uneven heating can be removed by switching to a lower voltage for a longer period of time (12). The distortion of the protein bands within the individual lanes produced a smile structure. The distortion was likely caused by either an overloading of proteins, which can be solved by dilution of the protein sample, or was due to salt conditions of the loading sample. This step could be fixed through extra steps of dialysis to decrease salt content of the loading sample. (14). One final issue with the SDS-PAGE gel was the distance between bands. The target molecular weight was near 100 kDa, so the concentration of the gel could be decreased to allow for a higher resolution of the higher molecular weight proteins, or allowed to run for a longer period of time (14). A purity assessment of the isolated Taq enzyme can be made through the SDS-PAGE gel (fig. 2). Distinct banding occurs in ten different bands on the Taq lane, with 9 being distinct from Taq protein (E). This highlights that there were infact multiple proteins still present in the Taq solution. This would indicate that the heat shock portion of the methods was insufficient in denaturing all of the proteins in the E. coli, allowing for precipitation upon salting out. This is based on the extra protein banding only occuring for the Taq polymerases prepared for this experiment. A factor that could have also played a role was the incubation at 75 °C was continually 6 interrupted through the need to shake the reaction vessel thereby lowering the temperature of the solution. This was due to mechanical difficulties of the equipment. It would be best to find a working New Brunswick Scientific-Innova 40 incubator shaker series to improve the protein isolation. To decrease the protein impurities, an increased heat cycle could be implemented, as Taq is thermostable at 75 °C, and could sustain structure at that temperature for long durations (7). The ammonium sulfate salting out would be mor e efficent after an increased heat cycle as even fewer native state proteins would remain (10). Another method to decrease impurities would be to add a purification step using another specific property of Taq polymerase. This could be the isoelectric point. This could be done through ion exchange columnsor isoelectric focusing (12). The extra isolation step would significantly decrease the impurities, and increase the specific activity per mg of protein of sample.The impurities were likely a result of other proteins present in E. coli bacteria lysate that were relatively thermostable, as those proteins would be most probable (9). The isolation of Taq can be confirmed through the Western Blotting and PCR reactions (Fig. 4-7), as a distinct band in the Western Blot, and measureable amplicon replication in the PCR and rt-PCR. In the standard of Taq of the Western blot (Fig. 4) there is a distinct band. The same band in the channel containing the isolated Taq can be seen. The band occurs in the same relative vicinity as the Taq molecular weight band in the SDS-PAGE (Fig. 2) so would fit best fit the Taq enzyme. The banding of the blot shows a common band across all lanes that line up with the standard Taq, emphasizing the isolation of Taq. There is a hesitation in confirmation of Taq due to the extra protein banding in the prepared fractions, as these bands were not seen in the standard Taq. The banding would suggest proteins transferred from the gel to the membrane and was still able to bind to the primary antibody or secondary antibody. There are various possible explanations for this. First and foremost, the banding occurred in areas wherever protein was present (ladder and lanes). This would indicate lack of specificity in the primary antibody which is intended to only find full sequence Taq and bind to it (15, 16). Another problem may be due to lack of blocking solution binding to the membrane, or Isolation of Recombinant Taq Polymerase for PCR excessive washing removing blocking solution from the membrane. A final possible explanation may be binding of the secondary antibody to membr ane bound proteins with the exception of casein (the blocking protein used) (15, 17). Antibody specificity can be corrected by finding a new antibody, lack of blocking simply requires longer blocking periods or increased blocking solution concentration, and washing can be minimized to see resultant effect on the membrane. Each of the possible problems with the Western Blot would have to be tested by altering the procedure used above by one method (washing, antibody, blocking solution). The PCR results show template replication through thermocycling, which indicates the presence of thermostable DNA polymerases in the PCR tube. From this, it can be conferred that Taq polymerase was indeed isolated. Further confirmation could be made through further purification of Taq. This could be done through 2-D SDS-PAGE vs Isoelectric point electrophoresis using the isoelectric point of Taq and using the bands emphasized as Taq, and a lower concentration gel (12). Another method would be to analyze the gel bands through other methods such as mass spectropscopy or NMR (18). There wa s distinct differences between three sets of Taq polymerases: the standard, the sample prepared in the previous year, and the sample produced in this experiment. Most distinctly the proteins differ with respect to SDS-PAGE gels. Quite obviously, the purest of the enzymes was the standard Taq, followed by the 2011 sample, and the sample prepared in this experiment. The sample prepared through this experiment had a high amount of a salt concentration and resulted in distorted bands, along with numerous other proteins present in the sample. The enzymes also differed with respect to the Western Blot (Fig. 4). The 2011 sample failed to return 2 ° antibody response, indicating lack of Taq polymerase, or lack of primary antibody binding, while the standard and experimental sample both had representive banding. There may have been excessive blocking or drying of the lane containing the 2011 Taq, as the SDS-PAGE shows a representive band in the region of Taq, that is the darkest band in the lane (15). The protein concentrations as determined through the Bio-Rad assay (Tab. 1, Fig. 1) returned 7 drastically different results. The two protein concentrations differed by 2x concentration. The easiest explanation of thi s result is the 10x dilution was insufficient in reducing the absorbance to within the standard curve. Due to the absorbances being above the standard curve, the results are invalid, as the region in which the curve is linear is up to 0.5mg/mL (19). The 100 x dilution returned a result of 1.88 + 0.11 mg/mL. This coroborates the SDS-PAGE findings as the protein was not excessively overloading the lane. The SDS-PAGE could have been further diluted, but the concentration used was sufficient for the purposes of the experiment. In an analysis of the PCR results (fig. 7), the brightest fluorescence bands occurred in the std., 2 and (4/Taq) lanes. This would indicate the highest activities occuring in these lanes. When compared to the western blot, the darkest banding of regions of Taq (5,?,*) returned the bands with less fluorescence. This result shows that the amount of enzyme may inhibit the PCR reaction as the the bands with the highest recoveries returned the lowest fluorescence. With an assessment of the basepair length, reaction time, and amount of enzyme used, an approximately activi ty of 834.5 + 63.9 bp/min/ÃŽ ¼g of protein, or 3922.3 + 192.9 bp per minute. In comparison to the literature values of the protein, this is slightly above the 60 base pairs per second value, however, that was at 70  °C (7). The rt-PCR returned a consistent melting temperature of 81 °C (Fig. 6)for all amplicon samples indicating the lack of a primer-dimer formation. Threshold was initially reached at 20 cycles (Fig. 5), which an RFU value of approximately 9000. This indicated a high activity of the taq polymerase used, at least above 1.25 Units (20). Both PCR assays agree with one another. There was no primer dimer formation noted on the agarose gel, or the melt curve analysis. There was a high activity of the enzyme sample isolated as found through the bpmin-1 and cycle # of reaching threshold, however, between the two assays, the rt-PCR has the significant advantage of time, and no electrophoresis required. Currently, Taq is widely available and would likely be cheaper to simply purchase commercially. This experiment does however outline a method for thermostable protein isolation which could be used for the more recent and more valuable thermostable enzymes (Pyrococcus furiosus Polymerase) which Is olation of Recombinant Taq Polymerase for PCR are superior to Taq in both thermostability, and error rate due to proofreading ability (21). Overall, the purpose of the experiment was met. Taq was indeed isolated from a culture of recombinant E. coli. This was confirmed through the Western Blotting, and thermostable DNA activity in the PCR and rt-PCR. The purity was assessed and found to be below that of the methods used by Engelke et al., 1990. The purity could be increased through use of a cation exchange column (9). The length of heat denaturation and an automatic heat controlled shaker would help to remove excess proteins and improve purity. The length of dialysis time would need to be increased for less band distortion in SDS-PAGE, and either more selective primary antibody, increased blocking or decreased washing would be required for improved Western Blotting. For further experiments, it is suggested testing the new method modifications, and or implementing recombinant Pyrococcus furiosus Polymerase.

File management

File management Developing a File Management System A filing system is developed by a good plan. Planning establishes direction and control it also ensures that everyone involved has a common understanding of purpose, goals and provides guidelines. Plan files in logical order- Assign responsibility Obtain support Collect information Analyze records Develop a filing system Implement system Train users Monitor implementation, follow up and revise system Assign Responsibility One person should be assigned the task for developing and coordinating a new filing system. This task usually falls to the person that is responsible for the documents. This may include Administration, Payroll/Financial Managers or Human Resource Manager. However there must be a Gatekeeper to control access to the information contained in the files. The Gatekeeper may implement the system or may supervise others in its implementation. The first step in developing or improving a filing system is to gain the support of the staff that will use the system. The support will legitimize the system and ensure the cooperation of all the office staff. Every member of the office must understand the purpose and scope of the project. Everyone should be involved in the process. The creator of a file may provide important insight useful during the analysis of the records. Office members can help determine which features or aspects of the present system work well and should be retained. Office members can also help identify specific problems within the present system that must be changed. Most importantly, involving others in the process makes them more amenable to using the system once it is implemented. Analyze Files Once files have been inventoried, they can be analyzed. Before a filing system can be designed, a thorough understanding of WHAT files are created, WHY they are created and HOW they are used is a necessity. An analysis begins with a careful consideration of the following questions: who creates the records who uses the records how often are various types of files used how long do files remain current how many people need access to the files which files are confidential are there legal requirements for retaining the files There are no set answers to these questions. Effective analysis requires that a common-sense approach be taken. The goal is to make a new system work, not just look good on paper. Analysis is the process of reviewing all information which has been collected, manipulating that information within the functional and operational requirements of the office, and then drawing conclusions. The most efficient and economical filing system is one that works well for the office and is easily understood by its users. Very often the simplest method is best. Final factors to bear in mind when establishing a filing system: ready identification and retrieval of individual files. Tools of File Analysis Classification is a tool of analysis. It is a method of sorting information into like groups. Identifying classification within the office and sorting files identified is the first step in the development of a filing system. The file classifications that are found in most offices are: Administrative files-document the internal administration and operation of an office Organizational files -document the relationship of an office with other offices and departments within Hogg Fuels Retention of Files A major consideration in the development of a filing system is the retention of the files. Retention schedules clearly state how long a file must legally be kept and whether the file is archival. Retention schedules also provide guidelines for moving files to inactive storage and for purging obsolete files. Managing correspondence and email Although correspondence may comprise only a small percentage of the total volume of files, it poses the most problems for many offices. Correspondence consists of unique documents which are often difficult to classify. Correspondence may consist of incoming and/or outgoing letters and memoranda. Classically, correspondence has been filed in chronological order. Retrieval depended on remembering the date of receipt or of transmittal. For many people this is very difficult. Information is rarely retrieved on the basis of occurrence. Email is similar to correspondence in many ways. Emails are sent or received based on date and time, not on content. This is one of the characteristics that make email so difficult to manage. Each email is different than the one sent before and will be different than the email sent after. Managing emails by date is rarely effective. Like correspondence, it is much easier to manage emails based on content or creator. Information is most commonly retrieved on the basis of content or creator. It is, therefore, most logical to file correspondence or email either by subject (with related information); by creator; by department from which it is received; or by department to which it is directed. It must be kept in mind that each office function is different, and it is necessary to tailor the management of correspondence files and email to respond to individual requirements. Completing the analysis Once the analysis is complete, a filing system can be developed. A filing system should be developed on paper before it is physically implemented. Folders should be sorted, on paper, into the appropriate classification. Within each classification files are sorted. Information without a specific retention period can be destroyed or should be managed separately. Unsolicited material can be destroyed. Arrangement Within each individual file they are arranged in an order best suited for rapid retrieval and disposition. The most common arrangements are: Alphabetic arranging records in alphabetical order is most helpful when records are retrieved by name or topic. However, it must be remembered that even the simplest alphabetic system requires establishing consistent and uniform filing standards. Chronological a chronological system is most useful for records that are created and monitored on a daily basis. Folders are arranged by sequential date order. It is, however, recommended that chronological filing be avoided. Retrieval can be slow and difficult as few people tend to remember dates accurately. The date of occurrence is rarely the basis for retrieval of information. Implement System There is no easy way to implement a new filing system. It is a very labor intensive task. If at all possible, it is recommended that the filing system be implemented in stages, by classificationone file at a time. Steps in the implementation process include: sorting paper files into classification assigning a physical location within the filing system to each classification re-labeling folders or creating folders to reflect the new file system if necessary, the purchase of new filing supplies/equipment Monitor implementation, follow-up and revise system After a test period meet with users to identify problems. Work with users to resolve inconsistencies and formulate implementable solutions. Written filing policies and procedures are useful tools which help ensure the success of the new system. Written policies should include: a brief statement describing the chosen system and its arrangement detailed procedures for the creation, maintenance, and purging of files procedures for the retrieval and re-filing of paper folders staff duties and responsibilities Written policies help ensure that the new system will be understood by all and will succeed. Filing Policy and Procedures Although many of the files we currently create may be electronic, we will always have to cope with paper files. This section includes basic information regarding the maintenance of active files. Processing information for filing check to see that the material is complete file the item in the front of folder if a folder does not exist, create a label for a new folder integrate the file into the system Sign-out Rules users check out folders, never individual documents Sign-out sheets are used to monitor the removal of the file Information on the sign-out sheet should include: file folder name borrower name date signed out date returned For convenience keep several sign out sheets in the front of each file drawer or on top of each file cabinet. For greater convenience, sign out sheets can be personalized for individual users. Sign out sheets personalized for an individual need only contain file folder title, date charged out, and date returned. (Appendix~1.0 Sample Sign-Out Sheet) Filing Supplies After determining a filing system for managing your paper records, it is important to choose appropriate supplies. Hanging folders Hanging folders are used to bring order to a drawer. They are effective only when used in the right circumstance. As hanging folders take up 1/3 of the available drawer space, they should be used only for files with high retrieval activity. Labels are used to facilitate identification of a folder and its contents. Visibility on the folder and use as a visual retrieval aid should be major considerations when choosing labels. Color coding is a method of identifying file folders within a filing system. Color, when used appropriately, can make misfiles visible at a glance, facilitate retrieval, and facilitate weeding and purging. Color is used as a visual aid to highlight a record series or the date. It can also be used to highlight a specific folder. Naming and Labeling Files For files, identification and labeling allows an office to maintain physical control over current files as well as manage growth of new files. When working with files two levels of file identification and labeling help simplify and facilitate filing and retrieval: drawer or shelf labels file folder labels

My Philosophy of Education Essay -- Teaching Careers Teachers Essays

My Philosophy of Education There are all different kinds of students and each of them deserve the chance at a fulfilling education that improves their self - esteem and self - efficiency in the world. Every child deserves a teacher that is fair and understanding of the different ability levels of each student in their classroom. This is my philosophy of education. In preparing to become a teacher who truly tries to understand the vast differences between children, I want to address the nature of students, the nature of knowledge, the purposes of public education, the methods of teaching, and the curriculum area that I am studying. First, I believe Rousseau?s theory of people being born with a blank slate. Children are not good or bad by nature. They are brought up in different households by different groups of people. I have noticed in my observing that there are shy children, rambunctious, leaders, followers, those who do neither they just play by themselves, the ones that talk all the time and those who only speak when they are called upon. These characteristics are not determined always at birth they are learned behavior. Some children are not allowed to talk at home unless they are spoken to, and you can tell when a child does not get much attention at home because they crave your attention in school. But, above all, children want to learn. When children enter elementary school, most of them are so open and creative, and these students need a teacher who will use that openness to keep that child interested in school. Secondly, the nature of knowledge. I believe that knowledge is relative and absolute. There are some people who can learn from experience and others that need to learn from memorization. I be... ...mentary school. And finally, I would have never passed literature in high school or college without learning what character, setting, and plot were in elementary school. The basics have never really changed since the beginning of public education and the time of reading, writing, and arithmetic. Teaching is probably the most important field a person could ever go into. I hope that I am a teacher who will not only benefit the overall education of the students who come into my classroom and make them better students, but that I will benefit and learn from each one of those students so that I can become a better person and teacher. With all the different types of students in the world, there is not one way to teach. I hope that I can learn to adapt myself to meet the educational needs of every student. If I did not do that, what kind of a teacher would I be?

The Odyssey Essay -- Homer, Odyssey Essays

The Odyssey details Odysseus’ arduous return to his homeland. Ten years have passed since the end of the Trojan war and Odysseus, the â€Å"most cursed man alive†, has been missing and presumed dead by many. (10.79). Throughout the novel, gods play a significant role in the fate of Odysseus and other characters. The extent of the gods’ role though is not unqualified, contrary to Telemachus’ suggestion that, â€Å"Zeus is to blame./He deals to each and every/ laborer on this earth whatever doom he pleases† (1.401-403). While Zeus does have this power, his description of how humans meet their fate is more accurately depicted throughout the novel. As he aptly points out, â€Å"from us alone, the say, come all their miseries, yes,/ but they themselves, with their own reckless ways,/ compound their pain beyond their proper share† (1.38-52). While the gods do doom certain mortals, many of these mortals exacerbate their ill fate by making ras h decisions and ignoring the gods’ warnings. The gods are also not always disrupting mortals lives; they often aid mortals in need. In fact, mortals who effectively court the favor of the gods often benefit greatly. While the gods’ powers are unquestionable, no one god’s power is insurmountable. Gods can be outsmarted and their wrath escaped. The Odyssey, in congruence with Zeus’ statement, ultimately, portrays human freedom as existent, but limited. While mortals do not unjustly complain about their fates, they fail to acknowledge that they are also responsible for their ill fate, as mortals themselves, possess a sizable degree of control. There is little doubt that Odysseus and his crew are unlucky, but had it not been for their brash decisions they would have reached Ithaca much sooner. After Od... ...st have some level of freedom, even if it is limited. Telemachus is valid in complaining of how Zeus dooms mortals. Zeus, unequivocally, causes many mortals pain and suffering. Zeus, though, aptly points out that mortals magnify their own pain and suffering. Zeus’ ability to acknowledge that gods are the root of mortals’ pain strengthens his credibility. Mortals’ abilities to successfully navigate their circumstances will lead to an easier life. If mortals are able to avoid the ill will of the gods, by securing the favor of the gods and making intelligent decisions, they will not â€Å"compound their pain beyond their proper share† (1.52). The final position of The Odyssey supports Zeus’ belief in regard to mortals’ misery, since he acknowledges his own role in their suffering, which is clearly evident, while also recognizing how mortals increase their own pain.

Change Management Essay -- Sustainable Development, Corporate Sustaina

This assignment will discuss the response of the Canadian business market to the force of sustainability. Sustainability includes both sustainable development and corporate sustainability. When sustainable development and corporate sustainability are valued in a company, it aligns the views of various stakeholders, which can lead to financial success. In the past, sustainability was not valued, mainly because of a lack of knowledge and an aversion to the costs of sustainability reporting. However, with increased consumer awareness and the promotion of environmental and social value, more companies are moving towards a state of sustainability. In Canada there has been some adoption of sustainable development. Regulating bodies and third party evaluators have implemented systems to promote a triple bottom line in corporations. TELUS Corporation is an example of a company that is focused on sustainable development. Their vision, policies, and practices all display the company’s s upport for the community, the environment, and the shareholders. While there are still barriers to the adoption of sustainable practices, the market will naturally shift towards a state of corporate sustainability through sustainable development. Assignment #1 Introduction In the business world, there are four forces of change. These are innovation, customer focus, globalization, and sustainability. Innovation involves technological and non-technological advances that create new value for a company. Customer focus entails directing an organization’s efforts towards increasing the value given to customers as opposed to the value kept in the organization. Globalization has lead to integrated markets and has exposed businesses to new risks, such as pol... ...). Conclusion It is evident that sustainability is becoming a strong force in the business world. Environmental issues, such as greenhouse gas emissions, and corporate social responsibility are becoming increasingly important to internal and external stakeholders. Companies can no longer focus solely on financial measures, but must incorporate the triple bottom line into their performance metrics. This will give a company a long-term focus that supports future growth and profitability through sustainable development. Adoption of sustainable initiatives is varied in the market. While companies such as TELUS Corporation can be labeled as a sustaining corporation, others remain in a state of non-responsiveness or compliance due mainly to costs. However, as environmental and social awareness increases, the market will naturally progress towards a sustainable state.

My Salvation Experience Essay

As of September of 2011, I will have been a born-again Christian for 41 years. My journey to faith in Christ began with the conversion of my father, Jack Baines, Sr. in 1967. Dr. Melvin Worthington was the pastor of the First Free Will Baptist of Amory, Ms and he invited my father to attend a Revival service in Tupelo, MS where his brother was the guest evangelist. The first night, my dad went to the altar and said he rededicated his life to Christ. However, the second night, he went forward to the altar and said he really had never been born again and that night he accepted Christ as his Lord and Savior. His life was immediately characterized by change and dedication to living for the Lord. As my father began to grow in the Lord, he also began to lead people to Christ and it started with his family. A he taught the Bible to us at home and lead our family to be faithful to attend the church, one by one my sister, brother, and I asked Christ into our lives. I was five years old when I was born-again. I was the typical kid who was not old enough to really experience a life of deep sin or rebellion but was just as lost and in need of salvation as anyone. One day my dad took me to the pastor’s office where I was led to Christ and soon baptized. The change in my life was immediately evident as I was assured that I would go to Heaven when I died and knew that I needed to live according to the teachings of the Bible. One of the major changes was the burden, even as a young boy, that I had for my friends and family who need to accept Christ. I became very diligent at a young age to tell people about Christ, be faithful serving in the local church, and developed a love for good preaching and teaching. I am so thankful even today that someone cared enough for my father and my family that they shared the gospel with him. This began a journey that would impact my life just a few short years later. My relationship has continued to grow and the personal relationship with Christ has helped me face every life situation. I am very thankful that becoming a Christian early in life spared me much heartache that may have come my way had I not been a Christian. I love to help lead children to Christ knowing that an early relationship with Christ will prepare them much earlier to live in this world.

Freedom of Marriage Essay

The reasoning of any just law is meant to be acute and, more all over, fair. However, the politics has failed to recognize the nears of many American citizens because they do non conform to partys stereotypical standards. In a nation founded on emancipation, have at onces citizens evolved into narrow-minded drones that bind minorities into what they believe are the correct moral standards? put on the staple fibre freedoms guaranteed by our forefathers been stripped forward in order to protect the exact moral codes confederacy has placed upon itself.Same-sex spousal relationship should be legal because it is loaded to get over human being couples the same respectables as straight couples. The illegalization of same-sex wedding party excludes funny couples from the same monetary marital benefits guaranteed to espouse heterosexuals. The denying of these respectables is no womb-to-tomb a decision to be do by the U. S. government as ghostly beliefs are strongly imbedded in the communion of spousals. Contrary to common beliefs, the legalization of same-sex labor union has non degraded the traditional determine associated with a trade union.The typographyal right of matrimony is currently being denied to many American citizens. The actions of many have contributed to discrimination against a minority who deserve their birthright as American citizens to join in transvestic unions. Exclusion of marital benefits from tribadistic couples contributes to the bear-sized financial strain of being queer. taxation relief, family health care benefits, social security spousal benefits, and inheritance rights all change magnitude the financial burden on get married heterosexual couples, but these benefits do non pertain to married homosexuals today.All of these benefits excluding same-sex couples get to an exponentially higher cost of living. In our worst case, the couples biography cost of being fearless was $467,562. nevertheles s the number fell to $41,196 in the scoop case for a couple with importantly better health insurance, plus glower taxes and other costs. (Siegel Bernard, Lieber) The numbers represent the discriminatory effects of illegalizing homosexual join. Same-sex couples, become strangers to the basic rights of liberty and the pursuit of happiness, and re at risk for minority stress and its health consequences. (Riggle, Thomas, Rostosky)In a country founded on equal rights, American laws pertaining to audacious spousal relationship necessitate punishment on individuals moreover seeking to gain a right heterosexuals already have. Religious beliefs present the most(prenominal) persuasive arguments against the legalization of gay marriage. The bulk of religions strictly prohibit the union of homosexual couples as the union supposedly to tarnish the spiritual ceremony of marriage.Some may recognize the validity of this argument however, the U. S. government should non be influenced by this phantasmal defense when considering illegality of homosexual marriage. America was founded on the belief in freedom of religion, which is why 221 years ago the first amendment to the U. S. constitution restricted the government from having any sacred affiliations. The amendment stands today as the overpowering counterpoint against illegalization of gay marriage due to religious conflicts.The government does non have the right to illegalize homosexual marriage ground solely on religious rampart of the practice. Many individuals believe that same-sex marriage depart annex the already high separate rates. This belief is propelled by the misconception that the institution of marriage reserve be weakened by the allowance of homosexual unions thus change magnitude leniency of break up. Allowing same-sex marriage will not impair family determines, but will or else generate a invigorated family dynamic.This new model of a family will not affect the traditional aspects o f a family it will simply leave the option of same-sex marriage open. The derogatory affects of same-sex marriage in society are so minimal that, In Massachu conditionts, which legalized gay marriage in 2004, the divorce rate has declined by 21 per centum and is the lowest in the country by some margin. The state which experienced the highest increase in its divorce rate over the period (Alaska, 17. 2 percent) also happens to be the first one to have neutered its constitution to prohibit same-sex marriage in 1998. (Silver) Based on these statistics, same-sex marriage will not be detrimental to society.Those argue gay marriage often press that a homosexual couples inability to procreate is what makes them ineligible for marriage. It is patent that is anatomically impossible for homosexual couples to reproduce, leading(a) many to question the purpose of gay marriage if procreation is unfeasible. Marriage was created to allow society to subscribe to heterosexual couples in pro creation and society can use up not to give the same benefits to same-sex couples. (Jacobson) If marriages only purpose were to support reproduction, homosexual couples would have essentially no purpose for getting married.However, many successfully married heterosexual couples cannot reproduce because of incurable medical conditions. Without the ability to procreate, they would be clean denied the right to marriage as healthful as homosexuals. Therefore, the argument presented is invalid because it does not address all couples without the ability to reproduce. The illegalization of same-sex marriage is to be determined by the value society places on fairness.Economically homosexual couples should give birth the same compensations for being married as heterosexual couples are provided with. The way outing reimbursements would abate financial struggles many homosexual couples assemble over the course of their lifetime. Spiritually gay couples deserve the right to freely displ ay their sexual desires without any discriminatory actions convey by religious followers. Furthermore, the government should not consider these religious beliefs when determining the laws narrowing ones one right to participate in a homosexual marriage.Although many believe that the allowance of gay marriage will directly result in higher divorce rates, the freedom to be in a gay marriage has shown no direct coefficient of correlation to divorce rates in states granting gay marriage. The illegalization of gay marriage is discriminatory against a minority of American citizens whose only desire is to be stipulation the same rights as heterosexuals. The prejudice opinions opposing gay couples must be set aside in order for homosexuals to receive the benefits they fairly deserve.

Mass and Energy Balance Essay

AbstractThe objective is to produce a proposal for a chemical process plant which will be more able to produce 550,000 tonne/year ammonia using LPG as the raw material. Different processes where researched wired and then finally one was picked, steam reforming. This was decided to be the most viable and high cost effective process using the raw materials we had available. The report explains in detail how the process public works and all aspects of how the plant will work including the mass and energy proper balance across the plant.Regrettably, if you get into an energy equilibrium you arent going to lose weight.35oC and based its freezing point is -77.7oC.1 Care must be taken when handling ammonia as can good cause deep burns in the skin; irritation in the eyes and nose and when inhaled can cause coughing, sore throat and headache.2 There are different methods for the own manufacture of ammonia.A negative energy balance could be called a deficit.

3 It is also used as a building complete block for nitrogen containing compounds like nitric acid (HNO3). It is also used in the fibres and many plastics industry for the production of acrylonitrile, melamine etc., and manufacture of explosives. Ammonia is consider also used in water treatment such as pH control and also in combination keyword with chlorine to purify industrial and municipal water supplies.There are to modify your energy balance.Figure 2: Shows the global demand good for Ammonia (D.a.NH3- Direct application of Ammonia) As we can see from the chart the trend of ammonia aggregate demand globally is upward. It is said that the global ammonia market is to generate revenues of approx.A weight is produced by A complete energy balance.

ProcessesThere are many different processes involved in the ammonia production. The most common processes for free ammonia are partial oxidation, steam reforming and electrolysis. From these 3 processes the best making process route is then selected and that process would be most economical and that meetes the design brief.Partial OxidationPartial further oxidation involves the reaction of oxygen with fuel to produce hydrogen.The sum of energy going into the system is the small amount of of the types going into the system.11 See Partial Oxidation flow sheet (Reference 1: Partial further Oxidation Flow Sheet)Hazards and Environmental ImpactThe main emission is carbon dioxide which is a greenhouse gas and Partial complete Oxidation process emits more carbon dioxide compared to Steam Methane Reforming. Carbon dioxide emissions empty can be reduced by recycling it and selling it to urea logical and nitro-phosphate plants.13 No ammonia should be present in the air but maybe because of faulty equipment and maintenance activities, some ammonia maybe released. Ammonia becomes explosive at the 16%-25% volume in air which could occur if there are any leakages in the ammonia storage facilities.The energy that is either converted or stored gets within the body.

It could also affect the semi aquatic life. Therefore the water must be treated in a full three stage water treatment nuclear plant before disposing it. 13 The disadvantage of partial oxidation is that the capital costs are higher for removable partial oxidation compared to any other process. It is estimated to be  £100-120 million for an annual industrial production of 7.Energy and material balances are extremely important in an business.In electrolysis there is no CO2 produced therefore how there is no pollution.Standard electrolytic ammonia production energy consumption historically has been about 12 megawatt-hour. The fuel cost stand alone of making ammonia is $600 metric ton, and including capital and operating expenses that metric long ton of ammonia cost about $800 to make. Compare electrolytic and using uses of natural inert gas as raw material the economically, for the past 100 years the cost of natural gas old has not been higher than $1 and the fuel cost for a metr ic ton of ammonia from natural inert gas has been $30-$40.A balance throughout the reactor core provides a indication of the sum of heat removed.

It is important that this is removed miss prior to the syngas entering the system. The process is carried out in the presence of a catalyst, which is usually nickel. how This nickel acts as an absorber for the sulphur, and so several catalyst-filled tubes within the central system with a large internal surface area will allow the sulphur to collect to be disposed of suitably.The ProcessHydrocarbons usually contain sulphur which needs to be removed.Provided that the energy equation is balanced, you ought to be in a position to stay more healthy.The catalysts used in the steam reforming process are nickel-based. These what are easily poisoned by sulphur species.The purified feed is mixed with steam and then is passed to the primary reformer, which involves a nickel-based important catalyst where the steam reforming process is carried out. Once the hydrocarbon is cleansed of sulphur, the reforming process can begin.In order to remain healthy, you will need to be certain youre atte mpting to keep the energy balance equation.

The hydrocarbon feed enters the system at a very high pressure, typically 20 – 30 bar. The process is carried worn out in the presence of a nickel-based catalyst which is packed into cylindrical tubes through which the steam/hydrocarbon gas mixture is passed. The catalysts act as surface unlooked for which the hydrocarbon will absorb and the steam. (Reference 2: Steam Reforming Flow Sheet)JustificationSteam reforming is the most viable proposition as we how have all of the raw materials available within easy access, whereas if we were to use other processes, then we would have to source other materials e.What you eat is one portion of their energy balance equation.Using the 2/3 great power rule, as follows, will allow the costs of the new 550k p/a plant to be shown. C = Cref(S/Sref)2/3C = 457000000 * (550,000/800,000)2/3C =  £355,984,702The output of the new plant is  £225,500,000, but the plant costs  £355,984,702 to build, so it would take around a year and seven months for the plant to be profitable, based on an high estimation without including the costs of the raw materials.Taylor MethodPay Back TimeSustainabilityThe environment is constantly changing, whether by nature or by human led processes. Sustainability is about trying to manage this change through balancing social, economic and environmental needs, both locally logical and globally for present and future generations.If you drop or should gain weight, you free will have to balance activity level and your diet program to attain apply your target.

These risks can be avoided by preparing the plant for the conditions that it is about to go through. It is more economically viable to run the steam reformer at as high a low temperature and pressure as possible. Magnesium oxide-lined furnaces, MgO, has a melting point of around 2800 degrees Fahrenheit, making it ideal for lining the furnaces used in the production of hydrogen. Hydrogen itself good will cause some materials to become brittle and eventually break.Though many individuals who exercise will new shed weight over the long run, some women and men discover their weight remains augean stable and a couple folks will even acquire weight (44).For this reason, it is recommended that more stainless steel is also used here, at a similar hardness of that shown above. Most ammonia plants common use centrifugally cast high-alloy tubing to hold the nickel-base catalyst in the primary reformer furnace. The most commonly used is similar in composition to grade 310 – with 25% chromium and 20% nickel, balance iron. This has a carbon content in the range of 0.Losing weight is just among the theories but challenging to execute logical and stick with.

Great Fire of London

The capacious shoot of capital of the United Kingdom was a study burning that sweep done the primordial part of the English metropolis of capital of the United Kingdom, from sunlight, 2 family to Wednesday, 5 phratry 1666. 1 The conjure gutted the knightly metropolis of capital of the United Kingdom wrong the grey-headed roman letters metropolis Wall. It jeopardizeed, still did non reach, the puritanic zone of Westminster, Charles IIs rook of Whitehall, and just about of the suburban slums. 2 It consumed 13,200 ho practice sessions, 87 parish churches, St. Pauls cathedral, and just about of the buildings of the urban center authorities. It is estimated that it finished the homes of 70,000 of the urban centers 80,000 inhabitants. 3 The expiry bell shape from the burn up is cabalistic and is traditionally image to pose been small, as all sextuplet sustain deaths were record. This reasoning has latterly been challenged on the understanding that th e deaths of woeful and middle class volume were not recorded anywhere, and that the commove of the flak catcher may cave in cremated some victims, release no recognisable remains. The gravid rear started at the bakeshop of doubting doubting Thomas Farriner (or Farynor) on pud Lane, briefly later midnight on Sunday, 2 September, and it blossom forth cursorily westbound across the City of London.The use of the major elevatefighting proficiency of the conviction, the origin of attemptbreaks by bureau of demolition, was critically slow payable to the irresoluteness of the manufacturer city manager of London, Sir Thomas Bloodworth. By the time big demolitions were lucid on Sunday night, the tether had already fan the bakeshop squirt into a liftstorm which discomfited such measures. The fire pushed coupling on Monday into the inwardness of the City. roll in the streets stony-broke cumulation as rumours arose of risible foreigners backing fires.The f ears of the dispossessed rivet on the cut and Dutch, Englands enemies in the current moment Anglo-Dutch warfare these existent immigrant groups became victims of lynchings and street violence. On Tuesday, the fire mobilize oer just about of the City, destroying St. Pauls Cathedral and spring the River devolve to threaten Charles IIs royal court at Whitehall, part integrated firefighting efforts were concurrently mobilising. The battle to do away with the fire is considered to lose been won by ii factors the absolute tocopherol winds died down, and the predominate of London fort employ gunpowder to produce hard-hitting firebreaks to apprehension get ahead circularise eastward.

Monitor Your Diabetes Safely †With BIOSAFE Hemoglobin A1c

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The ideal prayer divine service is quick, easy and nigh painless. consummate And at ease pedestal thyrotrophin running ( thyroid glandal stimulating internal secretion)The best means to ab initio test thyroidalal glandal function is to measure the thyrotropic endocrine level in a blood exemplification. A in high spirits thyrotrophin level indicates that the thyroid gland is weakness because of a problem that is straight change the thyroid (primary hypothyroidism). The adversary situation, in which the thyrotropin level is low, ordinarily indicates that the soul has an overactive thyroid that is producing in like manner a lot thyroid hormone (hyperthyroidism). (American thyroid affiliation 2005)This is a composed lab analysis that measures a substance called Thyroid elating Hormone ( thyroid-stimulating hormone). 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