Elizabeth Bishop’s Poetry Free Essays

Elizabeth Bishop poses interesting questions delivered by means of a unique style. Do you agree? Focus on themes and stylistic features. In my opinion, Elizabeth Bishop has a unique style of asking interesting questions. We will write a custom essay sample on Elizabeth Bishop’s Poetry or any similar topic only for you Order Now Bishop invites us along on the journey with her. She does this by her â€Å"painterly eye† which she has been praised for. In her poems she takes the ordinary and turns it into the extraordinary. As a reader, I wonder why she goes into so much detail. There is a story behind each of her poems. Her poems â€Å"First Death in Nova Scotia† and â€Å"In the Waiting Room† are about childhood experiences. She uses great detail in her poems and we feel like we are apart of it. This can be clearly seen in Bishop’s poem â€Å"The Fish†. â€Å"The Fish† is an example of where Bishop turns something so plain into the extraordinary. She takes fishing and turns it into a seventy-six-line poem. This poem recalls a time when Bishop went fish in a rented boat. Bishop makes a clear statement in the opening line of the poem, â€Å"I caught a tremendous fish†. The adjective tremendous is very effective, I feel. In the first four lines, Bishop stated how she caught a huge fish and stared at it beside her boat. She didn’t haul the fish into her boat. I question why she didn’t bring it straight on board. Bishop’s delight in catching the fish soon gives way to an emotional involvement with the fish. She compares his eyes to her own and she notes that the irises are â€Å"backed and packed with tarnished tinfoil†. The image is emphasized by assonance and alliteration. It was a big personal achievement to catch the huge fish. Bishop began to enjoy her triumph. It was a big moment for her. She imagined that her feeling of victory filled up the rented boat. Meanwhile, the big fish was still partly in the water. Then she did something unusual. She released the fish she had caught: ‘And I let the fish go’. I wonder why she had mercy on the fish and decided to let it go. â€Å"Filling Station† is another clear example of Bishop turning the ordinary into the extraordinary. In this poem Bishop is writing about a family petrol station. The voice in the poem is that of an outsider. The compound words â€Å"oil-soaked† and â€Å"oil-permeated† give us a clear vision of this petrol station. I wonder why Bishop is there in the first place. We become fascinated with the place. In verse two, the speaker sees the family. The image of everything covered in oil is continued. Alliteration is used to describe the sons, â€Å"several quick and saucy and greasy sons assist†, this suggests they have an oily appearance. The speaker begins to wonder if anyone lives here, â€Å"Do they live in the station? †. Bishop looks for and finds evidence of the female touch in verses four and five. We begin to see that there is beauty and love in the most unlikely places. In this male-dominated world, there is care to attention and detail with the mention of â€Å"daisy stitch†. In the final verse the repetition of â€Å"somebody† highlights the importance of the mother. The poem ends with the assurance that everybody is loved and worthy of love. Bishop recalls a childhood experience in her poem â€Å"In the Waiting Room†. This poem is similar to â€Å"First Death in Nova Scotia† as both have a theme of childhood innocence in them†. Perhaps the most immediately striking feature of Bishop’s work is its child narrator describing the seemingly innocuous event of waiting at the dentist’s office while her aunt is in the patient’s room. In this setting, the memory revolves around the narrator reading a  National Geographic  magazine. Bishop writes in uncomplicated, declarative language like â€Å"It was winter. It got dark / early. † that mirrors her age at the time. The poem takes an interesting direction as the child-speaker sees herself as a young woman: â€Å"What took me / completely by surprise / was that it was me: / my voice, in my mouth†. Aunt Consuelo’s cry becomes the speaker’s own cry. The woman and the girl merge into one in a surreal leap of the imagination â€Å"I – we – were falling, falling†. This poem makes us question what it means to be a woman. In â€Å"First Death in Nova Scotia† Bishop presents an extraordinarily vivid memory of a disturbing personal experience. It is winter in Nova Scotia. The dead child has been laid out in a â€Å"cold, cold parlour†. As in â€Å"In the Waiting Room† the voice in this poem is that of a child-speaker. How to cite Elizabeth Bishop’s Poetry, Papers

Literature Review Water Consumption

Question: Discuss about theLiterature Reviewfor Water Consumption. Answer: Introduction Background and Overview of Water Consumption In the previous studies, it has been identified that the water consumption in different countries has increased. However, supply of drinking water in the countries is not enough for meeting the demand for the drinking water (Xu et al., 2009). The water shortage occurred in different countries because of the carelessness of the people. Many times, the people misuse the drinking water. The water pollution is the other reason behind the shortage of drinking water. Due to the high water pollution, the quality of the drinking water has decreased, which causes different diseases. Due to the shortage of drinking water, the people in different countries especially in UAE are suffering much. The solutions of this problem are desalination and reuse of grey water. Definition of Water Consumption Water consumption can be defined as the consumption of fresh water, which is taken from the ground or other surface sources of water for temporarily or permanently (Khan, Badr Al-Zubaidy, 2014). The cost of desalination and reuse of grey water is much high, which may affect the economy of the countries. Thesis The increased rate of water consumption is a big problem for the countries and this problem must be solved as soon as possible. Theme 1: The water consumption is playing rule in different aspects In the words of Khan, Badr and Al-Zubaidy (2014), water is the primary source of life. Alkend and Omar (2013) stated that water is the basic need of any life. The function of water can be identified in several areas like, houses, schools, factories and other places. The households use water in order to perform their day-to-day activities like, cooking, washing, gardening, drinking and others. In schools and colleges, water is used for drinking, cleaning and washing purposes. Different factories use water in order to run their production and at the same time to supply the water to the workers for drinking and cleaning purposes. Xu et al., (2009) stated that water is also important for the growth of the plants. Water helps the plants in the photosynthesis process. This means, water helps the plants getting their foods, which is the main source of growth. On the other side, the consumption or use of water has direct effect or relationship with the economies of different countries. In the countries, where water shortage exists, requires desalination or reuse technology to maintain the smooth flow of water supply to the people, plants and animals. Maria Botros has stated in her article Solutions to tackle water scarcity in the UAE that the cost of implementing and processing the desalination and reuse technologies cost much, which can increase the cost of the economy. If the cost in the economy increases, the income level automatically decreases. Theme 2: The Effects of the Water Consumption in Several Aspects The over consumption of water has impacted the life and organisms. Over water consumption created fresh water shortages in different countries, especially in UAE countries. Due to this, the governments in the countries are requiring implementation of advanced technologies, which are costly (Gulfnews.com, 2017). However, the water shortage problem has not been solved completely. In many UAE countries, people are not getting enough drinking water and due to that many diseases are taking place. As per the statistics, every day, 5000 children die due to the shortage of water or due to drinking low quality water. The worlds statistics have shown that in last 50 years, the freshwater consumption has increased by 64000 billion liters. The statistics have also shown that to produce one liter of bio-fuel, near about 4000 litres of water is required (Worldometers.info, 2017). Over use of water creates water shortage, which has direct impact on the climate and environment. The level of global warming has increased due the water shortage. At the same time, the water shortage is also causing threats to the plants. The quality of water is decreasing, which is also affecting the plants as well as people and other animals (Md. Bader, 2011). As the agriculture in the countries depends on water, the water shortage or bad quality of water is also negatively affecting the agriculture sector. Theme 3: The Solutions to Reduce Water Consumption and How Control it In order to solve the water shortage problem, the governments as well as people in the countries require to come forward. The water supply authorizes of the countries can reduce the water consumption by setting a specific consumption standard for each household and factory (Gulfnews.com, 2017). The governments can implement specific water consumption laws in order to control the water consumption. The government can also warn the people in the country to be more careful about the water usage. Along with these, the government can also implement the desalination and reuse technologies in order to solve the problem. These technologies will help to produce more fresh water or drinking water. Conclusion In this study, it has been identified that the level of water consumption has increased much in the present scenario. Due to the over consumption of water, many problems have occurred like, global warming, critical diseases and shortage of drinking water. In order to solve these problems, the governments can set water consumption standards and can implement desalination and reuse technologies. If the water consumption is reduced, then the countries may enjoy several benefits. If the water consumption in some countries is reduced, then the other countries, where the people face water scarcity, can get required amount of drinking water. The people can survive properly. The reduction in the water consumption will also help reducing the global warming and maintaining the standard of natural environment. Reference List: Alkend, R. R., Omar, S. (2013). Total Bacterial Quantification in Homes' Water Tanks.International Journal of Applied Biotechnology and Biochemistry,3(1), 51-74. Gulfnews.com. (2017). Dubai News, Abu Dhabi News, UAE News and International News from GulfNews.com plus Gold rates, sport scores, city guides, prayer times, Dubai financial data, weather forecasts and more. GulfNews. Retrieved 4 February 2017, from https://gulfnews.com/ Khan, S. A., Badr, A., Al-Zubaidy, S. N. (2014, March). Valuation of Chilled Water Condensate through Air Conditioning to Diminish Water Scarcity. InInternational Journal of Engineering Research and Technology(Vol. 3, No. 3 (March-2014)). ESRSA Publications. Md. Bader, F. (2011). The Effects of Different Faucet Aerators on Bacterial Growth and Water Quality, 1(1), 1-92 Worldometers.info. (2017). Worldometers. Retrieved 4 February 2017, from https://www.worldometers.info/ Xu, P., Wang, G., Cath, T., Drewes, J. E., Ruetten, J. (2009). Consider the Pros and Cons of Desalination (PDF).Opflow,35(3), 10-13.

Johnsonville Sausage Essay Example

Johnsonville Sausage Essay Johnsonville Sausage – Quality Improvement through Change Situation Post 1980, Ralph Stayer began to observe decline in quality due to inattention of the workers as well as ill-maintenance of the equipment in the production unit. With the help of Mr. Lee Thayer, Stayer has planned to implement a major change in the company’s philosophy. Transition from old philosophy purged Stayer’s responsibilities of decision making by decentralizing the power of decision making. The new philosophy was implemented to bring out changes in various departments of the company viz. management structure, personnel, compensation, manufacturing and also systems and controls. According to Thayer’s new philosophy, performance of the personnel and requirement of guidance to personnel to achieve their objectives were the key elements. Management structure was broken down by hiring experienced personnel for the new superior level management to oversee finance, marketing and operation s functions. Stayer emphasized people development as the core element of the new philosophy. Changes in personnel include development of list of responsibilities by the workers themselves and eliminating the performance review process. These changes in personnel function led changes in compensation with the implementation of â€Å"company performance share† program which increased individual responsibility of workers. However this transition was not in agreement among workers while some are excited about it. In terms of manufacturing changes, lead persons were installed at various departments, eliminating supervisors, to seek information and provide necessary measures to train and improve capacities of workers under designated group. Responsibilities of lead person also include development of budget planning and production requirement planning. Job switching and alternating works were practiced to enrich workers’ capabilities and enhance performance and also job satisfaction. System and controls changes included individuals to develop and be accountable for their own budgets and forecasts and also allow them to analyze performance data. In addition, subordinates were needed to self-evaluate their performance to respective managers. Problem The main issue of the case is the resistance shown by the personnel towards the company’s philosophy transition. According to Stayer, upon implementation of new philosophy, workers at all level were expected to embrace the change. We will write a custom essay sample on Johnsonville Sausage specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on Johnsonville Sausage specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on Johnsonville Sausage specifically for you FOR ONLY $16.38 $13.9/page Hire Writer Instead, the company faced issues related to the final goal of the implementation, unsuccessful organization structural changes, clash of responsibilities among the workers, participation of less number of employees in the company decision making process and finally lack of high job satisfaction. However, these issues were expected to expire once the transition is complete. Palmer Sausage offered its consolidated business to Johnsonville but the issue here was whether Stayer could handle an expanded Palmer contract. Stayer examined the situation by understanding that in order to run the contract the company would require to recruit and train a big set of people along with existing people acquiring new skills and retain high quality on both the in-house and Palmer products. In order to achieve this, Johnsonville needs to run the production six or seven days a week for more than a year until its new plant was ready. On the other hand, the company may need to run the business at risk i. e. , if Palmer canceled the contract with 30-day prior notice leaves Johnsonville not only with massive layoffs and but also huge inventory with no market for it. Analysis As the year 1980 folded, Stayer recognizes to restructure his management structure for the growing company. Stayer also realized that there was decline in the quality of the products and employees were also demotivated to work. Later on, in collaboration with Thayer Lee, Stayer has planned to implement new philosophy which is focused on performance and helps people accomplish their objectives through well-defined job descriptions, clear understanding on performance standards, and providing the required resources. Johnsonville’s new values are based on deep moral commitment to the individual. The main aim of the transition is to augment people capacities, rather than the business. Stayer changed view of his autocratic leadership to a role as a leader delegating his subordinates rather than directing them. Furthermore, the new philosophy also motivated each worker to commit to the company’s betterment. Upon full transition, Stayer expects to create an atmosphere of constant learning and development. The transition also supports creativity, encourages responsibility, and empowers employees to develop solutions rather than simply follow orders. Over the period of time, the company’s organizational structure was transformed in support to the new culture of worker empowerment. Autocratic management was decentralized in order to become more democratic. Conventional management position were replaced by lead persons and their teams who controlled their own compensation, hiring, firing, quality control, and near-and long-term company goals. Several ground-breaking solutions were instigated and evaluated and each member of the company was involved for the company’s success. Moreover, a rewarding compensation system was implemented to retain employees and hone their skills and performance. Over-the-time, Stayer had achieved a complete organizational transformation successfully. In terms of Palmer’s offer, Stayer has two recommendations – either he can accept or decline to take over the Palmer’s business. If Stayer had taken over the business, they might have the advantages of the opportunity for growth and additional revenue. On this regard, private label business offers a 25% return on assets. Contrariwise, the deal clashes with Johnsonville’s recent business strategy which prevents the company to push private label business over 15%. The company could compete for capital with the rest of the business once it crosses 15%. In addition to this, the company, while in contract with Palmer, is producing at huge capacity utilization. If the deal is on then the company might need to execute two long shifts for six or seven days a week in order to process the new business. Recommendation At this stand-off point, Johnsonville could be advised to accept Palmer’s deal which help the former to tap the revenue and growth opportunities. Stayer’s Johnsonville should continue to implement its new philosophy in a way that it does not affect the previous work-culture. During the agreement, Stayer should continue to involve employees in the decision making process to accept the private label business and planning to handle the additional workload as well. Apart from this, Stayer is required to motivate his teams to meet for evaluating staffing requirements, production and quality goals. With the involvement of workers and assigning responsibility for the important decisions, Stayer will help secure their buy-in for tackling the challenges ahead.

Revisiting Wether, Incidence and Different Than

Revisiting Wether, Incidence and Different Than Revisiting Wether, Incidence and Different Than Revisiting Wether, Incidence and Different Than By Maeve Maddox wether/whether In researching the recent song lyrics post, I came across a comment written by a high school sophomore. (For the information of non-American readers, a high school sophomore is 15 or 16 years of age.) The student said she was writing a research paper on the influence of song lyrics. I certainly hope she looks up the spelling of the conjunction whether before she finishes her assignment; she used it four times in her comment, each time spelling it wether. wether (noun): a castrated ram. whether (conjunction): one use is to introduce an indirect alternative question expressing doubt or choice between alternatives. More at â€Å"Wether, Weather, Whether.† incident/incidence NPR (National Public Radio) announcers are a rich source of nonstandard English. On a recent morning I listened to Sam Sanders report on a pediatrician who prescribes exercise to his overweight patients. One of the doctor’s techniques is to encourage patients to visit local parks. Sanders mentioned that safety is a concern. He said that one of the parks, Kingman Island, â€Å"had 30 incidences of violent crime over the past year.† The erroneous use of incidences for incidents was cleaned up in the transcript, but it can be heard in the audio (3:33). incident (noun): something that occurs. incidence (noun): the range or scope of a thing; the extent of its influence or effects. For example, â€Å"The incidence of poverty among  the aged has consistently been higher than for any other age group in the United States.† More at: †It’s Not the Ox-Bow Incidence† different from/different than/different to A reader asks, â€Å"Is the correct usage ‘different to’ or ‘different from’? Different to seems very common (almost universal), but surely the essence of difference is separation, not convergence, so isn’t ‘different from’ correct?† This question comes up frequently, often with angry attacks on speakers who use the â€Å"wrong† phrase. Of the three, â€Å"different from† is by far the winner on the Ngram Viewer. â€Å"Different to† is heard more frequently in Britain than in the United States. â€Å"Different than† has its American defenders, but the AP Stylebook comes down firmly for â€Å"from, not than.† The Chicago Manual of Style is less dogmatic, but does say, â€Å"The phrasing different from is generally preferable to different than.† More at †Different from, Different to, Different than.† Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Misused Words category, check our popular posts, or choose a related post below:100 Words for Facial ExpressionsThe Possessive ApostropheRite, Write, Right, Wright

Strategy, Business Information & Analysis Essay

Strategy, Business Information & Analysis - Essay Example Sri Lanka is one of the few countries in the world, providing free universal healthcare, including both education and healthcare services (Workinsrilanka.lk, n.d.). Development of the healthcare industry is one of the major government priorities and for achieving its goals; the Government has initiated the National Health Master Plan (2006-2016). Government support has contributed significantly to the rapid growth and development of both public and private sectors, improved quality of healthcare services and human capital base in the industry (Workinsrilanka.lk, n.d.). As of 2012, Sri Lanka had 593 government hospitals and 197 private hospitals. The number of qualified doctors in total exceeded 17, 129 (approximately 1,187 persons per doctor) and the number of qualified nurses was 29, 871 (approximately 683 persons per nurse) (Workinsrilanka.lk, n.d.). In 2012, the total expenditure on health care both private and public was approximately US $89 per head, or 3.2% of GDP (The Economis t Intelligence Unit, 2014). Even though the level of expenditure on health is low, Sri Lanka illustrates significant positive trends: increase of life expectancy to 75 years, and fall of mortality rate among children t 9.6 cases per 1,000 births (The Economist Intelligence Unit, 2014). The quality of healthcare services provided in Sri Lanka is known around the world, as this country has high quality clinicians, adopts the latest innovations in medicine, science, and technology (Medicare, 2015). In order to have better understanding of the healthcare industry dynamic in Sri Lanka there was utilised the five forces model. There is a limited number of suppliers of medical devices, consumables and pharmaceutical items in Sri Lanka. In addition to limited availability of suppliers, Sri Lankan’s healthcare industry faces with a problem of limited availability of medical devices, consumables and pharmaceutical items often causing stock out situations. Limited number of

Business & Society ( Ethics & Stakeholder management) Essay

Business & Society ( Ethics & Stakeholder management) - Essay Example A.2: A CEO has to lead by example and John Mackey must be aware of this basic ethical tenet. Else, he would neither have taken the trouble to hide his identity nor chosen the Yahoo! Finance Stock Forum, to air his messages. Yet he persisted with this deception in spite of knowing the implications of his activity. The loss of prestige in the event of his being found out probably did not bother him too much. I would qualify his attitude as being ‘indifferent’ in setting high ethical standards for his company. A.3: A firm’s image is the sum total of its products and social responsibilities. Firms go to great lengths to reinforce their positive images in fields such as corporate governance, social responsibility, environmental compliance etc., in order to attract various stakeholders – employees, suppliers, consumers, investors and regulatory authorities. John Mackey’s activities are definitely harmful for the future of Whole Foods. The specific example of the difficulties faced in its attempts to acquire Wild Oats can be seen A.1: The reality of the situation (or, ‘what is’) is that my boss used official resources for his personal purpose, however insignificant it may be. This was not a one time, inadvertent practice, but indulged periodically with the full knowledge of himself and his subordinates. The normative (or, ‘what ought to be’) behavior demands that personal works are not carried out at the expense of the business. A.2: The boss’s practice is not ethical. He is trying to justify his action by rationalizing. Neither his length of service with the company nor the limit put by himself on the maximum postage at 60 cents, offer any justification for his conduct. If this issue is ignored, one has to answer the logical ends up to which such behavior can be accepted as ethical, in an infinite number of situations. A.3: If his argument of low pay being compensated by his unauthorized use of company resources

Putative S-Adenosyl Methionine Dependent Ironâ€Sulfur

Putative S-Adenosyl Methionine Dependent Iron–Sulfur Identification and Characterization of a Putative S-Adenosyl Methionine dependent Iron–Sulfur containing protein from  Methanococcus Jannaschii Qi Liu Research Proposal Dr. MishtuDey Dr. M. Lei Geng Dr. Leonard R. MacGillivray Dr. Amnon Kohen Dr. Daniel Quinn Introduction Biological methane formation is a microbial process that is catalyzed by microbes called methanogens, which belong to the third kingdom of life, the Archaea. Methane is formed at the final catalytic step by methyl-coenzyme M reductase (MCR), in which coenzyme B (CoBSH, N-7-mercaptoheptanoylthreonine) donates two electrons to reduce methyl-coenzyme M. MCR is a 300kDa enzyme, which is composed of three different subunits in an ÃŽ ±2ÃŽ ²2ÃŽ ³2 arrangement and contains 2 mol of the nickel tetrapyrrole coenzyme F430, which are buried deeply within the protein complex[1]. There are five modified amino acids were found out on the ÃŽ ±-subunit and near the active site of MCR from methanothermobacter marburgensis based on the X-ray crystallographic studies. They are 1-N-methylhistitine (HisÃŽ ±400), 5-(S)-methylarginine (ArgÃŽ ±271), 2-(S)-methylglutamine (GluÃŽ ±400), S-methylcysteine (CysÃŽ ±452), where the side chains are methylated and one thioglycine (GlyÃŽ ±445) forming a thiopeptide bo nd (Figure 1). Since the DNA sequence of the encoding MCR gene shows no unsusal condons or unusual codon usages at the positions in which the five modified amino acids were found, the modifications are introduced after translation [1]. According to vivo labeling experiments with L-(methyl-D3)-methionine, people found that the methyl groups from the modified amino acids are introduced biosynthetically from the methyl group of methionine by specific S-adenosylmethionine (SAM) dependent Figure1. Post Translational Modifications in MCR  enzymes. These methyl translational modifications are catalyzed by protein methylases that specifically recognize the amino acid sequences up and downstream of amino acid to be methylated. The genome of methanogens has many open reading frame predicted to be putative methyltransferases, which also agrees with the proposal that there are four different SAM-dependent protein methylases are involved in the post translation modification. A search of six kn own methanogenic genomes led to the identification of conserved open reading frame around the MCR gene cluster. Some methangenic archaea contain two MCR isoenzymes, designated MCR1 and MCR2. This conserved hypothetical protein is found around MCR1 in Methanococcus jannaschii and Methanobacterium thermobacter. The open reading frame from Methanococcus jannaschii, MJ0841 is annotated as a conserved hypothetical protein, which is found to be related to the radical SAM enzyme superfamily. The signature motif of SAM radical enzymes is three cysteine motif â€Å"CX3CX2C† (Figure 2), multiple sequence alignment of the putative gene from methanogens show the conserved CXGFCXXC, which is known to coordinate to [4Fe-4S] cluster. (Figure 3) Figure 2. Multiple Sequence Alignment of MJ0841 Homologues from Different Methanogens Figure 3. [4Fe–4S] cluster coordinated by three-cysteine motif CxxxCxxC. The fourth iron of the cluster interacts with a bound SAM. Specific Aims It is interesting and important to determine the function of this hypothetical protein. We speculate that this hypothetical protein, MJ0841, could be a possible candidate responsible for the post-translation modification of the methylated amino acids, or, is involved in the formation of the thioglycine in MCR. Research Plan, Results and Discussion Expression and Purification of MJ0841 MJ0841, a 1248bp gene, was cloned into pET28a vector. The resulting plasmid was transformed in E.coli BL21(DE3) cells for gene expression. In order to increase the iron content, MJ0841 was also co-expressed with PDB1282. Overnight cell culture grown at 37oC in Luria-Bertani (LB) medium containing both kanamycin (50ug/ml) and ampicillin (100ug/ml) was inoculated, in a 100-fold dilution, into Terrific Broth (TB) media aerobically at 37oC. FeCl3 was also added to a final concentration of 100uM to be as the iron content for iron-sulfur cluster during the growth when OD600 was around 0.3. Protein expression was then induced at OD600 of 0.6 to 0.7 with addition of Isopropyl ÃŽ ²-D-1-thiogalactopyranoside (IPTG) to final concentration of 0.5mM. After overnight incubation at 37oC around 18hours, the cells were harvested by centrifugation at 5000rpm for 30mins at 4oC, and stored at -80oC. The following procedures were all carried out in oxygen free environment at 20oC. Purification was conducted anaerobically at oxygen level always below 2.0ppm in anaerobic chamber. 23g Cell were moved into anaerobic chamber and resuspended in 120mL lysis buffer (50mM tris-HCl, 300mM NaCl, 5% glycerol pH 7.5), and Phenylmethanesulfonyl fluoride (PMSF) 1mM final concentration, 3 tablets of protease inhibitor, 2-mercaptomethanol 10mM) for 15mins. The cells were lysed by sonication for 15mins followed by centrifugation at 30,000 rpm for 40mins at 4oC to remove the cell debris. The supernatant was applied to a packed 15mL Ni-NTA resin column equilibrated with lysis buffer. The column was then washed with 5 column volume of wash buffer (50mM tris-HCl, 300mM NaCl, 10mM IMD, 5% glycerol, pH 7.5). The brownish protein was eluted by gradient elution with 5 column volume of wash buffer and 5 column volume elution buffer (50mM tris-HCl, 300mM NaCl, 200mM IMD, 5% glycerol, pH 7.5). SDS-PAGE was applied to analyze the desired clean protein fractions, which were then combined an d set for overnight dialysis with dialysis buffer (50mM tris-HCl, pH 7.5, 5% glycerol) with slow stirring. The pooled fractions were concentrated using an Amicon centrifugal filter with a 30kDa molecular weight cut off (MWCO). The collected protein was further purified with 20mL packed Q-sepharose column equilibrated with lysis buffer (50mM tris-HCl, 5% glycerol, pH 7.5). The column was then washed with 5 column volume of wash buffer (50mM tris-HCl, 200mM NaCl, 5% glycerol, pH 7.5). The brownish protein was eluted by gradient elution with 5 column volume of wash buffer and 5 column volume elution buffer (50mM tris-HCl, 700mM NaCl, 5% glycerol, pH 7.5). SDS-PAGE was applied to analyze the desired clean protein fractions, which were then combined for reconstitution (Figure 4). Figure 4. SDS-PAGE gel electrophoresis analysis of MJ0841 purification fractions Reconstitution of the [4Fe-4S] Cluster of MJ0841 in Vitro The above apo-protein (16uM, 40ml) was incubated with final concentration of 5mM DTT for 1h with slow stirring at room temperature. Then, cystein was added into the above solution by dropwise to reach 10 molar folds excess of protein. After 30mins incubation, 10 molar excess of Fe(NH4)2(SO4)2 was added slowly to provide enough iron content for iron sulfur cluster and incubated for 30mins. The resulting solution was incubated with 10 molar excess of Na2S finally, and the brownish protein solution changed to dark brown after adding Na2S. The above final protein solution was kept in 4oC overnight around 14 hours for building up enough [4Fe-4S] clusters. In order to remove the unbounded iron and sulfur, the overnight reconstituted protein was concentrated to 2.5 – 3ml of final volume and loaded onto a 5mL PT10 column equilibrated with lysis buffer (50mM tris-HCl, 5% glycerol, pH 7.5), and the final pure protein was combined. Reduction of [4Fe-4S] Cluster UV-vis spectroscopy was applied here for detecting the reduction of [4Fe-4S] cluster. A characteristic peak for [4Fe-4S] cluster was shown up near 412nm before reducing. Sodium Dithionite was used as the reducing agent and the stock solution was prepared freshly right before adding into the protein. 100 equivalents of sodium dithionite were mixed with concentrated protein, and the peak at 412nm was reduced (Figure 5). According to the results from UV-vis, the 4Fe-4S cluster was built up by reconstitution, also the [4Fe-4S]2+ was reduced to [4Fe-4S]+ by the reduction of sodium dithionite. Figure 5. UV-vis spectra of purified reconsituted MJ0841 Figure 6. EPR spectrum of as-isolated MJ0841 (blue trace) and purified reconstituted MJ0841 originating from a [3Fe-4S]+ cluster. reduced with 100 equiv sodium dithionite (red trace) EPR samples preparation and spectral collection EPR spectrums of as-isolated, oxidized and reduced form of MJ0841 are detected at 10K. All samples are prepared anaerobically. As-isolated protein was prepared with the protein without reconstitution, and exhibits a strong isotropic EPR signal, which is centered at g=2.01 same as the g=2.01 signal of the [3Fe-4S]+ cluster form (Figure 6). The oxidized form protein containing [4Fe-4S]2+ was prepared by injecting 200ul concentrated protein purified through PD10 column into EPR tube, and it normally shows silent EPR signal (Figure 7). Reduced form of as-isolated protein was performed by mixing with 100 equivalents of sodium dithionite with 200ul concentrated protein, which gives the reduction form of [4Fe-4S]+ cluster and shows the characteristic EPR signal with g factors of g=2.03 and g=1.92 (Figure 8). Figure 7. EPR spectrum of oxidized form of MJ0841. Figure 8. EPR spectrum of reduced form of MJ0841. SAM cleavage activity of MJ0841 The characteristic reaction for detecting radical SAM enzymes is reductive cleavage of SAM into S-adenosylhomocysteine (SAH) and 5’-deoxyadenosyl radical (5’dAdo). Assays were conducted under strict anaerobic conditions. The reaction assay contains the following: 50mM Tris-HCl, 5mM DTT, 5Mm sodium dithionite, 0.5mM SAM. Reactions were initiated by addition of SAM and carried out at 20oC for 20 hours. The control reaction was run under the same conditions as the above assay, but without presence of protein. Trifluoroacetic acid (TFA) (final concentration 5% v/v) was added to quench the reactions, which then were identified by HPLC analysis. HPLC analysis of SAM cleavage assay products After quenching by TFA, the reaction mixture was centrifuged and the supernatant was applied into HPLC analysis. 10ul of assay mixture was injected into C18 column, which had been pre-equilibrated with equilibrium buffer (40mM ammonium acetate, pH=6.2). Then the column was washed with a linear gradient from 0-50% acetonitrile for 30mins at room temperature to detect SAH and 5’dAdo. The UV-detector was set at wavelength 258nm, and the standard samples, SAM, SAH, and 5’dAdo, were run with the same condition as SAM cleavage reaction products. According to the retention time comparison between standard samples and products, formation of SAH and 5’dAdo were all detected via HPLC analysis when enzyme was present. In the absence of enzyme MJ0841, SAM was not consumed at all and there were no any products peak formed, which confirmed the SAM was cleaved by enzyme. The dark blue, red, light blue traces show the relative intensities of 5’dAdo, SAH, and SAM standards. The green trace shows the assay with the use of reconstituted MJ0841, and the SAH and 5’dAdo were both observed. The purple trace shows the control assay without MJ0841, and there was not any of 5’dAdo formed (Figure 9). Figure 9. HPLC analysis of the SAM cleavage assays Conclusions Future Work Initial results seem to show the [4Fe-4S] cluster and the enzyme activity. Since SAM was cleaved enzymatically, the products will be detected by mass spectroscopy to confirm the formation SAH and 5’dAdo. Furthermore, probable substrates of MJ0841 will be prepared, which should contain the amino acids that would be modified. The activity assays with substrates will be examined to detect the desired methylation reaction on substrates.