Secrets and Lies free essay sample

In this essay I would like to argue that in Secrets and Lies Mike Leigh is suggesting that secrets cause tensions in families and destroys relationships between family members. Firstly, I would like to consider that it is actually true according my own experience because there is been some lies and hidden secrets in my family as well and once they are exposed, it causes embarrassments to those whom are affected. Secondly, I believe secrets can form a negative and positive impact once they are exposed because, it can be a turning point where families consider reuniting and leave the past behind which obviously happens in Secrets and Lies. Sometimes it happens the other way around where Secrets and Lies destroy relationships between family members forcing them to abandon each other. In this paragraph I focus on how Secrets and Lies is compared to other movies based on a humanistic approach meaning that they introduce the nature of human behaviour in societies. We will write a custom essay sample on Secrets and Lies or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Mike Leigh assumes that,† The films that are made in most parts of the world arent independent films; theyre just films, really. Here â€Å"independent† films mean films made in spite of Hollywood. † (Miller) Leigh considers Secret and Lies as an independent movie in a sense that it contains realistic facts and truthfulness compared to those produced by filmmakers in Hollywood where they mostly focus on style, cinematic etc. Leigh is right in that case because, Secret and Lies looks like some sort of documentary yet it’s a film rather than stylish, cinematic which in my point of view reflects to what is called humanistic approach. It consists of simple elements that we all are related to and not too advanced with special effects or better graphics as movies made in Hollywood for instance. I strongly believe that it touches its viewers with its contents. According to Miller’s article, involving an interview with Mike Leigh, he claims that, †I’d say that what I do is work very, very thoroughly indeed and get the actors involved from the word go to create a world that really does exist, whether we point a camera at it or not. (Miller) To make it all work, Leigh let his actors carry on with improvising while acting in order to create an environment which he relates to reality. This reflects to Rosenbaum’s quote where he asks himself why â€Å"Why, then, have I liked Secrets and Lies so much that Ive been able to see it three times with pleasure, and found it as gripping the third time as the first? † (Rosenbaum) It is how Leigh let his actors create an environment he looking for, says the author! In my opinion, I believe Rosenbaum is amazed about Leigh’s work because, the producer reveals how he simply does to make his movies become â€Å"independent†. I assume that most producers scribble down a script and give it to their actors when it’s done and here Leigh does the opposite and the results prove to come out great. In this paragraph, I emphasize on the difference between Secrets and Lies in and Hollywood movies and what others say about his work. Rosenbaum argues that, †Set Secrets and Lies alongside most recent Hollywood pictures it looks like a masterpiece, but considered in relation to the best of Leighsay, Meantime or Grown-ups or High Hopesor the best of recent non-Hollywood films, it looks rather thin. (Rosenbaum) As the author puts it, Secrets and Lies don’t have enough detailed information to be classified as one of Leigh’s best movies because, he claims that it not one that sticks in one’s mind. I’m of two minds about Rosenbaum’s claim that Secrets and Lies isn’t one of Leigh best movies because its lack of extra material to be considered in that category. On the on e hand, I don’t think Secrets and Lies weren’t clarified enough for one to understand what is really going on. On the other hand, I believe Leigh didn’t want to include unnecessary scenes in order to make it too long and simply erase its taste for some audience to be able to watch more than once. Still I strongly believe that some find Secrets and Lies as their favourite depending on their interests. Especially for those who relate it to their own environments in real life. I’m going to look at the race issue which most viewers regard as a main concept and also was on how Mike Leigh himself, responds to this during an interview. When asked if Secrets and Lies emphases on a race aspect, Mike Leigh replies, it’s not what he focuses on and he also indicates that its only idiots who do think that way about the fact that Hortense is black and Cynthia is white because, some don’t even know that it is 1996 and people have learned to live with the race issue behind in some societies. In fact, he backs up his statement describing Hortense’s character stating that, â€Å"As you get to know her, you simply forget that shes black because you get to know her and it ceases to be an issue. (Miller) I see Leigh’s point on the race aspect here because, it is 1996 when Secret and Lies came out and at that time, some societies consists of mixed races where racism is limited to a minimum point. Perhaps he wanted to point out the fact that Secrets and Lies was about other issues rather than race. Hortense had something to do which was to find her birth mother and try to find out the truth. What happe ned to her real father? Cynthia did want to get further on that case other than finally accepting Hortense as her daughter. I assume she felt ashamed of whatever happened and wanted to keep that away from Hortense. Perhaps she didn’t want to upset Hortense by telling her the story nor herself. A quote from Boston Review states that, â€Å"In fact, a week before Leighs film opened in Boston, the local news was filled with stories about the reunion of just such a daughter and her birth mother-an Irish Catholic woman with two small children who had been raped in the early 1960s by a Black man. (Stone) This brings curiosity to my mind why the author gave this example? Is it because he was reflecting this to a race aspect or he wanted to lead the reader to point that Cynthia was raped and that’s how she got Hortense. In my opinion, Alan Stone tries to give an example in which he signalling the similarity of Secrets and Lies and what he read in the newspaper. I can’t really tell if the author what his message about this statement. Was it because he related what he read in the newspaper to Secrets and Lies about the reunion of white mother and the adopted daughter? Or what I mentioned earlier? I assume he focused on both terms. I going to look at the scenes in Secrets and Lies as they reflect to the paragraph above and why they are very good for not only me but also for those who think they familiarize to them in terms of what they have experienced or what they have seen in real life. Mike Leigh struggles to make sure the audience understands the film and he claims during an interview that, â€Å"I go to enough trouble to make sure that you do understand in the scenes where Morris is taking photographs the whole spectrum of society, the various kinds of people and various skin colours. † (Miller) He brings a closer picture of reality rather than stylish, cinematic so that people relate to it in comparison to movies made by gigantic industries such as Hollywood. He also reflects on how the environment looks like, people who live in it as shown when Morris takes pictures of different folks with different skin colours. In this case, it was filmed in London and according to me, that’s how the society looks like over there. Leigh ignores the race aspect in Secret and Lies because in this kind of society, people living in this environment have learned to live with it and it doesn’t affect many as it’s a mixed society with different ethnicities. In this paragraph, I focus on actors and their characters in Secrets and Lies in comparison to what most people go through in real life. Author Rosenbaum summarizes Secrets and Lies to a dysfunctional family seeking mutual acceptance in an example in the movie where he claims that, â€Å"Cynthia is a mess, barrelling her way through the story, but its her messiness that ultimately exposes the family wounds and therefore allows them to begin to heal. Secrets and Lies free essay sample In this essay I would like to argue that in Secrets and Lies Mike Leigh is suggesting that secrets cause tensions in families and destroys relationships between family members. Firstly, I would like to consider that it is actually true according my own experience because there is been some lies and hidden secrets in my family as well and once they are exposed, it causes embarrassments to those whom are affected. Secondly, I believe secrets can form a negative and positive impact once they are exposed because, it can be a turning point where families consider reuniting and leave the past behind which obviously happens in Secrets and Lies. Sometimes it happens the other way around where Secrets and Lies destroy relationships between family members forcing them to abandon each other. In this paragraph I focus on how Secrets and Lies is compared to other movies based on a humanistic approach meaning that they introduce the nature of human behaviour in societies. We will write a custom essay sample on Secrets and Lies or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Mike Leigh assumes that,† The films that are made in most parts of the world arent independent films; theyre just films, really. Here â€Å"independent† films mean films made in spite of Hollywood. † (Miller) Leigh considers Secret and Lies as an independent movie in a sense that it contains realistic facts and truthfulness compared to those produced by filmmakers in Hollywood where they mostly focus on style, cinematic etc. Leigh is right in that case because, Secret and Lies looks like some sort of documentary yet it’s a film rather than stylish, cinematic which in my point of view reflects to what is called humanistic approach. It consists of simple elements that we all are related to and not too advanced with special effects or better graphics as movies made in Hollywood for instance. I strongly believe that it touches its viewers with its contents. According to Miller’s article, involving an interview with Mike Leigh, he claims that, †I’d say that what I do is work very, very thoroughly indeed and get the actors involved from the word go to create a world that really does exist, whether we point a camera at it or not. (Miller) To make it all work, Leigh let his actors carry on with improvising while acting in order to create an environment which he relates to reality. This reflects to Rosenbaum’s quote where he asks himself why â€Å"Why, then, have I liked Secrets and Lies so much that Ive been able to see it three times with pleasure, and found it as gripping the third time as the first? † (Rosenbaum) It is how Leigh let his actors create an environment he looking for, says the author! In my opinion, I believe Rosenbaum is amazed about Leigh’s work because, the producer reveals how he simply does to make his movies become â€Å"independent†. I assume that most producers scribble down a script and give it to their actors when it’s done and here Leigh does the opposite and the results prove to come out great. In this paragraph, I emphasize on the difference between Secrets and Lies in and Hollywood movies and what others say about his work. Rosenbaum argues that, †Set Secrets and Lies alongside most recent Hollywood pictures it looks like a masterpiece, but considered in relation to the best of Leighsay, Meantime or Grown-ups or High Hopesor the best of recent non-Hollywood films, it looks rather thin. (Rosenbaum) As the author puts it, Secrets and Lies don’t have enough detailed information to be classified as one of Leigh’s best movies because, he claims that it not one that sticks in one’s mind. I’m of two minds about Rosenbaum’s claim that Secrets and Lies isn’t one of Leigh best movies because its lack of extra material to be considered in that category. On the on e hand, I don’t think Secrets and Lies weren’t clarified enough for one to understand what is really going on. On the other hand, I believe Leigh didn’t want to include unnecessary scenes in order to make it too long and simply erase its taste for some audience to be able to watch more than once. Still I strongly believe that some find Secrets and Lies as their favourite depending on their interests. Especially for those who relate it to their own environments in real life. I’m going to look at the race issue which most viewers regard as a main concept and also was on how Mike Leigh himself, responds to this during an interview. When asked if Secrets and Lies emphases on a race aspect, Mike Leigh replies, it’s not what he focuses on and he also indicates that its only idiots who do think that way about the fact that Hortense is black and Cynthia is white because, some don’t even know that it is 1996 and people have learned to live with the race issue behind in some societies. In fact, he backs up his statement describing Hortense’s character stating that, â€Å"As you get to know her, you simply forget that shes black because you get to know her and it ceases to be an issue. (Miller) I see Leigh’s point on the race aspect here because, it is 1996 when Secret and Lies came out and at that time, some societies consists of mixed races where racism is limited to a minimum point. Perhaps he wanted to point out the fact that Secrets and Lies was about other issues rather than race. Hortense had something to do which was to find her birth mother and try to find out the truth. What happe ned to her real father? Cynthia did want to get further on that case other than finally accepting Hortense as her daughter. I assume she felt ashamed of whatever happened and wanted to keep that away from Hortense. Perhaps she didn’t want to upset Hortense by telling her the story nor herself. A quote from Boston Review states that, â€Å"In fact, a week before Leighs film opened in Boston, the local news was filled with stories about the reunion of just such a daughter and her birth mother-an Irish Catholic woman with two small children who had been raped in the early 1960s by a Black man. (Stone) This brings curiosity to my mind why the author gave this example? Is it because he was reflecting this to a race aspect or he wanted to lead the reader to point that Cynthia was raped and that’s how she got Hortense. In my opinion, Alan Stone tries to give an example in which he signalling the similarity of Secrets and Lies and what he read in the newspaper. I can’t really tell if the author what his message about this statement. Was it because he related what he read in the newspaper to Secrets and Lies about the reunion of white mother and the adopted daughter? Or what I mentioned earlier? I assume he focused on both terms. I going to look at the scenes in Secrets and Lies as they reflect to the paragraph above and why they are very good for not only me but also for those who think they familiarize to them in terms of what they have experienced or what they have seen in real life. Mike Leigh struggles to make sure the audience understands the film and he claims during an interview that, â€Å"I go to enough trouble to make sure that you do understand in the scenes where Morris is taking photographs the whole spectrum of society, the various kinds of people and various skin colours. † (Miller) He brings a closer picture of reality rather than stylish, cinematic so that people relate to it in comparison to movies made by gigantic industries such as Hollywood. He also reflects on how the environment looks like, people who live in it as shown when Morris takes pictures of different folks with different skin colours. In this case, it was filmed in London and according to me, that’s how the society looks like over there. Leigh ignores the race aspect in Secret and Lies because in this kind of society, people living in this environment have learned to live with it and it doesn’t affect many as it’s a mixed society with different ethnicities. In this paragraph, I focus on actors and their characters in Secrets and Lies in comparison to what most people go through in real life. Author Rosenbaum summarizes Secrets and Lies to a dysfunctional family seeking mutual acceptance in an example in the movie where he claims that, â€Å"Cynthia is a mess, barrelling her way through the story, but its her messiness that ultimately exposes the family wounds and therefore allows them to begin to heal.

Free Essays on Women In WWII

Women’s Roles During WWII Women of today’s society hold positions of importance in their jobs. Women in World War II contributed to the women today having jobs. Before the war women were viewed as just housewives. World War II caused a change of the living status of American women. Women went from being housewives to working hard and being a major help during and after the war. Women took jobs to build airplanes for their husbands to use in the war. Five million women entered the work force, and made up 36 percent of the American labor force (Sinnot 13). Thousands of women took jobs in airplane factories. They assembled tens of thousands of military airplanes needed in the war effort (â€Å"American Women in WWII† 391). Some aircraft plants were as big as small towns, and the noise of production was deafening. The women’s muscles became sore, their hands were swollen, and they got backaches and headaches. Many women had trouble sleeping because the noise continued to throb throughout their bodies, even when they were at home. The women went through many pains, but did their best and made thousands of airplanes that helped in the war. Women also helped make ships to use in the war. Shipyards were accustomed to a male workforce, and were slow to accept the women workers. In 1943 there was a demand for 1.5 million workers, so the shipyards were forced to hire women (Sinnot 33). The women found that building a ship wasn’t that difficult because of their previous skills with assembling airplanes. They found a similarity between cutting out patters for clothes and for ships; the only difference was they were using steel rather than cloth. The women also excelled at welding, operating drill presses, grinders, and lathes. Shipyard workers were open to many dangers as they worked. They were exposed to the natural elements as they worked on the edge of the water. Some women had to climb high scaffoldings to complete thei... Free Essays on Women In WWII Free Essays on Women In WWII Women’s Roles During WWII Women of today’s society hold positions of importance in their jobs. Women in World War II contributed to the women today having jobs. Before the war women were viewed as just housewives. World War II caused a change of the living status of American women. Women went from being housewives to working hard and being a major help during and after the war. Women took jobs to build airplanes for their husbands to use in the war. Five million women entered the work force, and made up 36 percent of the American labor force (Sinnot 13). Thousands of women took jobs in airplane factories. They assembled tens of thousands of military airplanes needed in the war effort (â€Å"American Women in WWII† 391). Some aircraft plants were as big as small towns, and the noise of production was deafening. The women’s muscles became sore, their hands were swollen, and they got backaches and headaches. Many women had trouble sleeping because the noise continued to throb throughout their bodies, even when they were at home. The women went through many pains, but did their best and made thousands of airplanes that helped in the war. Women also helped make ships to use in the war. Shipyards were accustomed to a male workforce, and were slow to accept the women workers. In 1943 there was a demand for 1.5 million workers, so the shipyards were forced to hire women (Sinnot 33). The women found that building a ship wasn’t that difficult because of their previous skills with assembling airplanes. They found a similarity between cutting out patters for clothes and for ships; the only difference was they were using steel rather than cloth. The women also excelled at welding, operating drill presses, grinders, and lathes. Shipyard workers were open to many dangers as they worked. They were exposed to the natural elements as they worked on the edge of the water. Some women had to climb high scaffoldings to complete thei...

Lonmin Plc company analysis Essay Example | Topics and Well Written Essays - 2000 words

Lonmin Plc company analysis - Essay Example It is this affiliate formed to handle the mining business that eventually became renamed as Lonmin Plc in 1999 (4-traders, 2014:n.p.). The company operates in three major segments of the mining industry, where it has an exploration department, an evaluation department and a PMG operations department. Despite the fact that the company runs its operations in different countries in the Weste African region, most of the operations of the company, which entail the refinement and marketing of the metals after being mined in different regions is undertaken in the Bushveld facility in south Africa (Lonmin Plc, 2014:n.p.). The company is listed in three different stock markets, where its shares are trade in Germany, USA and in the UK. The market capitalization of the company as at 2014 was worth US$ 146.7 billion, which is equivalent to 95 billion British pounds (Guru Focus LLC, 2014:n.p.). The company has total 583.8 million outstanding shares, whose prices in the stock market ranges at betw een 160.70 and 163.64 British pounds (Guru Focus LLC, 2014:n.p). The company held a total of 38,2923 employees by the close of the financial year 2014, with 28,276 of the employees being employed full time, while 10,016 operated as part time employees. The company holds a considerable value of assets, both in its operating assets such as the machineries and equipment, as well as the stock of minerals in the various mines that the company has explored. While some of these minerals are already extracted and their value well verified, there is still some more whose value is still to be ascertained. The graph below presents the assets of the company that are held in minerals. In the year 2014, Lonmin Plc made a total sale of 441,684 ounces of Platinum, at a market price value of $1,537 per ounce, which contributed to a higher percentage of the company’s

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Green Fluorescent Protein (GFP) Mutants

Green Fluorescent Protein (GFP) Mutants GREEN FLUORESCENT PROTEIN (GFP) MUTANTS WITH ALTERED FLUORESCENCE INTENSITY AND EMMISSION SPECTRA Introduction: Now-a-days GFP is creating revolution in the field of science by its applications and properties.GFP is a stable protein extracted from the photo organs of the jellyfish Aequoria victoria by Shimomura et al in 1962. In 1992 the cloning of GFP has done. It is found in a variety of coelenterates (both hydrozoa and anthozoa) and it emits light by utilising energy from the Ca2+ activated photoprotein aequorin [1]. Energy transfer and the emission spectra of GFP can be affected by dimerization. Structure of GFP is cylindrical ÃŽ ²-can structure and has a chromophore located centrally. The chromophore is responsible for the fluorescence and the formation is independent of species but mainly depends on oxygen. GFP is a small protein and has been made up of 238 amino acids. Deletion of any seven amino acids either from C-terminus or N-terminus may result in the loss of fluorescence. Amino acid replacement is responsible for the change in colours of GFP. It has a molecular weight of 27 KDa an d has an absorption range at 488 nm and an emission range at 509 nm. It can accomplish high temperatures (65 ÌŠc) and basic PH range of 6-12 [2]. Increase in PH results in the decrease of fluorescence. Increase in the fluorescence and photo stability can be achieved by single point mutation at S65T. Fluorophore of the GFP is generated by using auto-catalytic process of continuous mechanisms. Visible excitation is one of the optical properties of GFP. Its derivatives are produced from the mutagenesis experiments like random and directed mutagenesis [3]. GFP is majorly used as a reporter in expressing genes. Protein and chromophore folding also constitutes as a major advantage of GFP. It can also be used in protein fusion by applying recombinant DNA technology. Aim of this research is to analyze properties of GFP by cloning, mutations, expression of proteins and purification. Objectives of this research are to sub-clone GFP into a vector and mutations are carried out by various mutagenesis experiments followed by expression of proteins and purification. Finally after purification properties are analyzed. Materials and methods: Initially DNA is isolated and GFPuv is sub-cloned into the pET28c vector from pET23 plasmid by speectrophotometric analysis. 5 µg of pET23GFPuv DNA is digested by using NdeI and HindIII restriction enzymes. And the digests are analysed by using Agarose gel electrophoresis. GFP fragment is extracted and purified using QIA quick gel extraction kit from QIAGEN and the recovered DNA is estimated. Recombinant protein is expressed in E.coli by ligation and transformation. To confirm the presence of GFP in the pET28c plasmid, colony PCR is used. Further mutagenesis experiments are carried out by designing oligonucleotide primers which will alter the spectral properties of the protein. Complementary primers containing same mutations are generated. Mutagenic primers are prepared with a melting temperature of ≠¥ 78 ºC, length between 25 and 45 bases and primers longer than 45 bases are generally used. Introduction and identification of mutations within GFPuv gene: Mutations are created in the GFPuv insert by site-directed mutagenesis Site-directed mutagenesis: 5 µl 10 x PCR buffer 5 µl 20 mM dNTP mixes 15 ng GFPuv-pET28c template DNA 125ng oligonucleotide primer F+ 125ng oligonucleotide primer R+ 2 µl 25mM MgSo4 32 µl sterile water 1 µl KOD hot start polymerase (1U/ µl) * All the above are added to 0.2ml PCR tubes and incubated in a PCR machine for 24 cycles: 94 ºC 30s 94 ºC 30s 55 ºC 1min 68 ºC 4min 20s 68 ºC 10 min * Reaction is then kept on ice for 2 min and 1 µl (1U) of Dpn1 is added and incubated for 60 min at 37 ºC Alignment of amino acid sequences is carried out using: http://www.ebi.ac.uk/Tools/clustalw2/index.html Product of site-directed mutagenesis (pET28c DNA) is transformed into XL-1 supercompetent cells. Transformed colonies are extracted using QIAprep Mini prep kit Qiagen [5]. Concentration and purity can be checked by using Agarose gel electrophoresis. For this 5 µl of plasmid preparation and 10U HindIII are digested at 37 ºC for 1h. Sequencing is then carried out by using 10 µl of DNA at a concentration of 50ng/ µl. E.coli BL21 (DE3) cells are prepared and are transformed into the pET28cGFPuv plasmid for expression Auto-induction method: Wild type protein (GFPuv) and the mutant protein are expressed in the expression vector [BL21 (DE3)] using auto-induction method. For this transformed colonies are inoculated into 3ml of LB-1D + antibiotic media and incubated at 37 ºC at 300 RPM for 6 hrs and O.D is taken. Inoculum is taken into the flask containing SB-5052 auto-induction medium along with antibiotic and incubated at 28 ºC at 300 RPM for 20 hrs. Cultures are then cooled for 1 hr. Total induced sample is prepared by taking 100 µl of cooling culture and 900 µl of SB-5052 media. Cells are then pelletized by centrifuging it with both total induced and non-induced samples and are resuspended in 100 µl of SDS-PAGE (sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis(PAGE)) sample buffer. 12% of polyacrylamide gel is prepared and the Soluble and insoluble samples are prepared by cell fractionation using BUGBUSTER. For this 1 µl of DNAase1 is used along with reagents. Cell suspension is then centrifuged at 13000rpm for 20mins. Supernatant is then used as soluble sample and insoluble is prepared by resuspending the pellet in 2ml binding buffer. SDS-PAGE buffer and binding buffer are added to the soluble and insoluble fractions. At 95 ºC all samples are heated for 5 min. Gel is then loaded as: Molecular weight standard-5 µl Uninduced sample 5 µl Induced total sample 5 µl Soluble sample 5 µl Gel has to run for 1 hr. And is transfered to a box of Coomassie blue stain. Western blotting: GFP protein presence can be verified using western blotting technique. Protein samples are first seperated by SDS-PAGE and are transferred to the nitrocellulose membrane. GFP bound to nitrocellulose membrane is then visualised by incubating the blot with His-probe which is linked to a HRP (horse radish peroxidase) enzyme (HisprobeTM-HRP solution is diluted to 1:5000 (1 µl in 5ml) ). His-tag of GFP protein is bound to probe. Blots are kept in TBST and probes and thus probes are visualised by chemiluminescence and these are photographed by chemiluminescent reader. Ni-NTA chromatography: His tagged GFP can be purified by Ni-NTA (nickel nitrilo triacetic acid) chromatography method. In this, sample of soluble protein is loaded on column packed agarose resin and the non-specific protein binding is removed by washing resin with buffer and is eluted by high concentrated imidazole of elution buffer. After elution the purification of protein is done by SDS-PAGE and Coomassie staining. The concentration of the protein is measured by Bradford assay. Fluorimetry and mass spectrometry: Properties of GFPuv protein are analysed by Fluorimetry and mass spectroscopy. Fluorimetry: In this wavelength and intensity of a molecule at specific wavelength are measured using fluorimeters. Perkin Elmer LS50B is the fluorimeter used to measure GFP. Quartz cuvettes are placed in a chamber to measure the concentration and intensity. The parameters set to measure GFP are: Excitation 440nm Emission 460-550nm Slit widths 4 and 4 Accumulation 5 20 µg/ml of protein concentration is used. The emission and excitor wavelengths are set at 509nm and 395nm. Mass spectrometry: GFPuv properties and molecular mass can be analysed by mass spectroscopy. The type of mass spectroscopy used here is electron spray ionization (ESI). ESI is a type of atmospheric pressure ionisation technique (API) which is used for biochemical analysis. JEOL HX110/HX110A equipped with electron ion source tandem mass spectrometers are used to analyse structural properties [7]. 1-10 pmol/ µl of protein concentration is used. Solvents used are: MeOH MeCN TFA During ionisation sample is dissolved in a solvent and is pumped through a steel capillary at a rate of 1 µl/min and voltage of 3 or 4KV is applied [8]. Ion current is amplified by the detector and the data system will record signals in the form of mass spectrum. RESULTS: Site-directed mutagenesis: Primers used for site directed mutagenesis (Mutant) Forward primer: 5-CACTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCC-3 Reverse primer: 5-GGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTG-3 Alignment of the amino acid sequence of the mutant with the GFPuv amino acid sequence GFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 mGFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 ************************************************************ GFPuv VTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120 mGFPuv VTTFSWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120 *****:****************************************************** Y66W GFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180 mGFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180 ************************************************************ GFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238 mGFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238 ********************************************************** Amino acid substitution: Y66W Belongs to Class 5, indole in chromophore (cyan fluorescent proteins) [6] eCFP CATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT 60 GFP ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT 57 ********************************************************* eCFP GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC 120 GFP GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC 117 ************************************************************ eCFP GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACA 180 GFP GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACA 177 ************************************************************ eCFP CTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAA 240 GFP CTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAA 237 ******************* ******************************** ****** Mutation eCFP CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCT 300 GFP CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCT 297 ************************************************************ eCFP TTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTT 360 GFP TTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTT 357 ************************************************************ eCFP GTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACAC 420 GFP GTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACAC 417 ************************************************************ eCFP AAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT 480 GFP AAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT 477 ************************************************************ eCFP GGAATCAAAGCT 492 GFP GGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCA 537 ************ eCFP GFP GACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCAT 597 eCFP GFP TACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTC 657 eCFP GFP CTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA 717 SDS-PAGE : Coomassie staining gel of (Sample 6): Marker GFP protein (soluble sample) Western blotting (Sample 11): Induced total sample GFP protein Ni-NTA chromatography: Fluorimetry: Mass spectrometry: Wild-type: Mutant: Discussion: Site-directed mutagenesis: In the site-directed mutagenesis mutation is carried out at the right place i.e., at 197 and 198 places. Tyrosine (TAT) is mutated to tryptophan (TGG), Y W. During this mutation protein undergoes many changes especially in the fluorescence. GFP turns into CFP (Cyan fluorescent protein) hence the light emitted will not be exactly green. CFP will have many peculiar features like rather than single excitation and emission peaks it possess double humping. Tag CFP possess some properties like: Structure monomer Molecular weight 27KDa Polypeptide length 239aa Fluorescence colour Cyan Maximum excitation 458nm Maximum emission 480nm Excitation coefficient 37000M-1 cm-1 Pka 4.7 Quantum yield 0.57 Brightness 21.1 Brightness is produced by the quantum yield and extinction coefficient. Dual colour visualisation of the protein expressed is enabled by the CFP. This has led to the Fluorescence Resonance Energy Development (FRET). SDS-PAGE: SDS-PAGE is carried out to separate proteins according to their electrophoretic mobility and experimental repeats will result in the purity assessment of the protein. Four wells are loaded with samples and 2 and 4 wells show protein result and as 1 and 3 wells dont contain protein they will be normal without any bands. Results shows that little amount of GFP has been observed in the insoluble and large amount of protein has been observed in the soluble sample. Uninduced sample cannot find GFP. Western-blotting: Western-blot is performed to make sure the presence of protein. Histidine tagged probe is added to confirm the protein present was GFP or not. pET28c plasmid contains T7 RNA polymerase promoter sequence. But this promoter is blocked by the repressor. Hence lactose containing medium is required for E.coli growth. Because lactose is used as carbon source, glucose is converted into allolactose. This allolactose will bind to repressor by unblocking promoter, and expresses GFP. Hence presence of glucose will result in Lac-I and is binds to the operator. Band observed in the blot is probably GFP and it has high level of intensity after induction. And it is necessary to confirm this by performing blotting technique using His probe to detect His tagged GFP. Bands are observed in the induced and soluble samples after performing western blotting confirming the presence of GFP. Ni-NTA chromatography: Purification of GFP can be done by Ni-NTA chromatography. For a recombinant protein the amino acid binding site with 6 or more His residues in a row acts as metal binding site. So hexa-his sequence is called as His-tag. His-tag sequence is present in the N-terminal of the target protein and is located in the promoter region adjacently to the GFP gene. During this process enzyme HRP is also bound to the probe. This HRP-probe will react with luminal 4 peroxidase buffer which is further used for purifying GFP by Ni-NTA chromatography. Purification by His-tagged GFP can be done by using several methods like Ni2+-poly (2 acetomidoacrylic acid) hydrogel. Displacement of GFP can be done by binding nickel to imidazole. This is mainly because of high affinity of nickel towards imidazole compared to GFP.Distinctive bands are supposed to observe in the elute1, elute 2 and also in the total soluble fraction. Bands formed states the presence of the GFP mutant. Absence of the bands states mutant a bsence. In the results bands are observed at the total induced and the soluble samples which state the protein presence. Even small amounts of bands are also observed in the insoluble sample. GFP protein produced in the induced total sample is approximately at 27KDa. Slight bands are observed in the insoluble sample as it may be because of some impurities. Finally the GFP protein has been detected. References: 1. Davenport D, Nichol JAC: Luminescence in Hydromedusae. Proceedings of the Royal Society, Series B 1955, 144:399-411 2. Ward. W., Prentice, H., Roth, A. Cody. C. and Reeeves.S.1982.Spectral perturbations of the Aequoria green fluorescent protein. Photochem. Photobiol. 35:803-808 3. Cormack, B. P., Valdivia, R. H., Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, In press 4. Darelle Thomson , Greg Smith. (2001).PCR-based plasmid vector construction for generation of recombinant viruses. Journal of Virological Methods 94, 7-14 5. Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615-619. 6. HEIM, R., PRASHER, D. C. TSIEN, R. Y. 1994. Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein. Proceedings of the National Academy of Sciences of the United States of America, 91, 12501-12504. 7. HARUKI NIWA, SATOSHI INOUYE et,al., Chemical nature of the light emitter of the Aequorea green fluorescent protein. Vol. 93, pp. 13617-13622, November 1996. Proc. Natl. Acad. Sci. USA. 8. â€Å"Mass Spectrometry: A Foundation Course†, K. Downard, Royal Society of Chemistry, UK, 2004.

An Analysis of the First Two Acts of The Tempest Essay -- The Tempest E

      The first two acts of The Tempest share a couple of inconsequential similarities and have some very contrasting differences. The similarities are, on the whole, superficial: Both acts consist of just two scenes and both acts are of a similar length. However, the similarities end there.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   The lengths of the scenes in each act differ somewhat: Act 1 has one extremely short scene and one very lengthy scene; Act 2 is composed of two scenes of similar length. Also, the tone of each act is very different, with Act 1 being serious and composed, whilst Act 2 is more comic, often descending into pure farce. Analysing one scene at a time will show just how different the two acts are.      Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Act 1, Scene 1 is entirely unlike the other three scenes in the first two acts. It is fast-paced, exciting and uncomplicated, allowing the audience to be drawn into the play before the more complex scenes begin. The only notable thing in this scene is the introduction of Gonzalo, who is shown to be a patient, calm and optimistic person. None of the other characters are defined particularly vividly, nor need they be - this scene is supposed to be action-oriented and too much characterisation and plotting would ruin the tense atmosphere.      Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Scene 2 contrasts greatly with the opening scene, being lengthy and dialogue-driven, with little action at all. This scene is very important to the rest of the play, as it sets up the main background to the main characters, as well as defining some of those same characters. It is Prospero's character which is explored the most in this scene; whilst he explains his history to Miranda, we begin to see the sort of person he is.       From his dialogue, one can... ...t to be highly entertaining. Whether one finds the first two acts entertaining or not, by the end of the second act many of the main characters and themes have already been well defined.    Works Cited and Consulted: Garnett, Richard. "Irving Shakespeare" The Tempest (and selected criticism). Charlotte Porter and Helen A. Clarke (eds.) Thomas Y. Crowell & Co. 1903. Knight, G. Wilson. "Shakespearian Superman" The Tempest D.J. Palmer (ed.) Macmillan & Co. 1968 Murray, J. Middleton. "Shakespeare's Dream" The Tempest D.J. Palmer (ed.) Macmillan & Co. 1968 Palmer, D.J. Shakespeare's Later Comedies: An Anthology of Modern Criticism. Harmondsworth, Penguin, 1971. Shakespeare, William. The Tempest. 1611. Ed. Stephen Orgel. New York: Oxford UP, 1994. Tillyard, E.M. "The Tragic Pattern" The Tempest D.J. Palmer (ed.) Macmillan & Co. 1968