Unit 6 Essay Example | Topics and Well Written Essays - 500 words - 1

Unit 6 - Essay ExampleLastly, David (2011) indicates that the domestic and the international accompaniments have not ceased to augment. The organizations are being bear upon by these changes to a great extent thus, making it difficult to achieve strategic evaluation. These aspects have made the set strategies impossible to implement. Even with the constant wait for information, strategic evaluation has not proven to be the best measure to curb the challenges that face the organizations today. David (2011) also says that these challenges have made strategic evaluation not assess its performance effectively, as well as take the right measures that would see the survival of the organizations. An analysis of the objectives of the strategic evaluators would be a perfect guide to limit these challenges.For successful contingency planning, Rao, Rao & Sivaramakrishna (2009) recommend seven meters. The first step involves identifying the favorable and unfit factors that could lead to the set strategy failing (Rao, Rao & Sivaramakrishna, 2009). Secondly, the authors indicate that is wise to denote the factors that would elicit effects on the set strategy (Rao, Rao & Sivaramakrishna, 2009). This is to mean when the contingent events are expected to take place. Thirdly, an assessment of the implications of every contingent event is good for contingency planning (Rao, Rao & Sivaramakrishna, 2009). This step would place the planners at a good position to estimate the expected benefits and losses that could occur from the selected events. The fourth step entails designing contingent plans, then evaluating the counter effects of the selected contingency plan so as to identify which plans are the best and which ones should be overlooked (Rao, Rao & Sivaramakrishna, 2009). The ordinal step involves determining the warning signals in an opportune time so as to counter them appropriately. Lastly, Rao, Rao & Sivaramakrishna (2009) indicate that developing plans to

Surrealism and psychoanalysis in modernist poetry Essay

Surrealism and psychoanalysis in modernist poem - Essay ExampleThe essay Surrealism and psychoanalysis in modernist poetry states the surrealism and psychoanalysis. duration, and finally modern periods through literary advancements. depth psychology involves a network of psychotherapeutic and psychological theories, originally explained by Sigmund Freud. Psychoanalysis has continuously expanded, revised and real in many different directions. In psychoanalysis there atomic number 18 many theoretical dimensions explaining the mental development of humans, hence this leads to many psychoanalysis treatments. Psychoanalysis is also used to refer to the study method of child development. Surrealism refers to cultural relocation which began in the beginning of 1920s the movement is best identified with its grotesque writings and artworks. Surrealism developed through the activities of Dada in the course of World War One, and Paris was the major venue for the movement. After the 1920s t he cultural movement went global, and further affected countries literature, visual arts, music, language and social theory. Psychoanalysis has certain tenets development of a person is through early childhood events in addition to inherited aspects of personality irrational motivations influence human experience, attitude and mannerisms irrational motivations are unconscious psychological resistance is a defense mechanism, which develops when motivations or drives are turned into awareness conflicts involving the conscious and the unconscious manifests through emotional disturbance.

Free trade Assignment Example | Topics and Well Written Essays - 250 words

part with trade - Assignment ExampleThe U.S. on the new(prenominal) side has increased the import of agricultural products to Canada and Mexico (Orme, 1996).The economic basis of trade is to exchange goods and work for currency. It will be abuse to common sense to bribe or rather get poor services in the name of saving our local industries from foreign competition yet we get poor services and goods. We would rather go for a specialist. Research shows American did well economically because they allowed assuage movement of goods across its neighbors. While Europe, because they are protecting themselves from their neighbors did not do quite well. I bought my nephew a play station made from China. China makes good toys for children. America produces good computers. We can, therefore, sell our computers to China as we buy their toys (Orme, 1996).Free trade has allowed Americans to import textiles from Mexico. My dress was made from Mexico. There are very few people wearing clothes m ade in America. There learn been slogans saying that buy America keep your job. However, it is costly to put trade restrictions in order to save the local industries. The taxpayers pay more than those that have free trade. The benefit of buying American goods is that jobs are saved (Orme, 1996).Trade restrictions should only apply in cases where there is a violation of preservation rules. For example, food or agricultural products posing a danger to human life due to use of hazardous chemicals should be restricted. International trade does not stop competition or even cause unemployment (Orme, 1996). International trade enables countries to be able to have a look at different types of goods and compare them to theirs. It will scrap local industries to produce better goods and services in order to maintain their

Liabilities of a Director Essay Example for Free

Liabilities of a Director EssayA comp whatsoever is usually established by individuals or Directors (officers included) in this in good example so as to belong it in appropriate manner in order to make maximum profits. For this to happen, it is for the ships company (Pandora Diamonds and Gems Pty Ltd in our case) to enter or make contr comes with outsiders identical Kaplan Bank Ltd and quad Solutions Pty Ltd as out-of-the-way(prenominal) as our case is concerned. The contr coifs made by the company with other outsiders be usually done by Directors who are, most often than not, act as representatives of that mentioned company and be able to be liable by the acts of its Directors as stated in s126. Directors acting on behalf of the company during any contracts are usually in a crucial purview of trust within that company, and therefore, the vast array of legal duties willing stretch out on their shoulders for retention that position. Due to this hot position, it is easier than not, for their powers to be abused in different ways. Directors have been kn proclaim to abuse their powers in office for their personal gain. For example, Directors have been held liable for inappropriately using companys funds in making their personal secret profits.In such cases, the corporate police force comes into play (the Common faithfulness and the Corporations Act 2001 (Cth)) and Directors or any other officer who recklessly breach specific duties and obligations should be prosecuted in the court of law. So for the purpose of liability, the common law and the Corporations Act 2001 (Cth) have been laid down so as to minimize the risk of wrongful behaviors by companys Directors. In this paper, I will discuss the responsibilities, liabilities and duties of a director(s) in a company and give examples how Directors of various companies have been accounted or held responsible for infringing Corporations Act 2001 (Cth) laws.Directors DutiesSince companies are usually es tablished and managed by directors and a number of officers, these directors have that ostensible authority, collectively, to represent that mentioned company and not as an individual Director. As I am going to talk intimately it below, some individual directors (Andrew and Brian in this case) have been known to enter in to some kind of contract with outsiders (other companies) and not collectively as board of directors, as allowed under s201J and s198D of the Corporations Act 2001 (Cth). Directors, in most cases, have contravened these sections of the Act because they have their own material personal interests outside the company and have disowned their duty to act honestly and in good faith to the best interest of the company as per Part 2D.1 of the Corporations Act 2001 (Cth). Directors can only rescind the civil or immoral penalties for breaching the Corporations Act 2001 (Cth) only if they fully understand their liabilities and duties (Sievers, 1997 and Cassidy, 2006).Some o f Corporations Acts laws, which directors need to be watchful for, in order to avoid contravening Corporations Act laws, are and not limited to1. Act in good faith and honest for the best interest of the company.2. By considering companys interests ahead of their own.3. Avoiding conflicts of duty and interest.4. Duty to avoid insolvent trading by the company1. Duty to act honestly and in good faith to the best interest of the companyAs far as s181 of Corporations Acts 2001 (Cth) is concerned, any director including Andrew and Brian in our case, have to act in good faith and in the best interest of the company (Pandora Diamonds and Gems Pty Ltd) (see Darval v labor union Sydney Brick and Tile Co (1988) 6 ACLC 154) and not for their personal intentional purposes. In our case, before resigning, Brian set up his own jewellery business so that he can engage in a lucrative business of supplying jewellery to his new friend Victor after resigning from Pandora Diamonds and Gems Pty Ltd. Bri an, under Corporations Act 2001 (Cth) s.184, whitethorn have infringed the statutory duties that are found in ss.181-183, and therefore liable for civil or criminal penalties as per Corporations Act laws (Sievers, 1997 and Cassidy, 2006).2. Duty not to improperly use the position of a director to gain any personal benefitsAs contained in s182 of the Corporations Act 2001 (Cth), directors must demonstrate their powers for the required purposes so that companies like Pandora Diamonds and Gems Pty Ltd for example can benefit and those directors who violate s180 of the Corporations Act law Sbe penalized or disqualified in the court of law as was illustrated in the case of Mills v. Mills (1938) 60 CLR 150 (Cassidy, 2006).In our case, the exercise of power by Brian to secure some personal advantage by starting his own jewellery business, is considered as an improper purpose because it is not within the purpose of benefiting Pandora Diamonds and Gems Pty Ltd, but to himself (see Mills v. Mills (1938) 60 CLR 150, Biala Pty Ltd v. Uallina Holdings (No 2) (1993) and ASIC v. Adler (2002)). Eventually, Brian will be responsible for any financial benefit he might have received as the head of Pandora Diamonds and Gems Pty Ltd (see Queensland mine Ltd v Hudson (1978) 18 ALR 1) (Cassidy, 2006, p. 251).3. Avoiding conflicts of duty and interest.The conflict of interest is a matter for all board of directors that does not only affect specific directors in the company. According to section 182 and 183 of the Corporations Act, any misuse of directors position just for the disadvantage of the company and for the benefit of the director is generally forbidden by the law (see Aberdeen Railway Co. v. Blaikie Bros (1854). If some sections like s191 to s195 of the Corporation Act is canvas further, it details some important rules on how Directors (Brian included) are supposed to disclose or declare to the board of directors, if there is any personal interest that is colligate to the affairs of the company. This is required so as to ensure that the honesty and integrity of directors are thoroughly observed (Cassidy, 2006).As far as our case is concerned, Brian may have violated s181 of the Corporations Act, for he did not declare or disclose to the board of directors that he has some interest of starting is own business as required under s191 (3) and he may be prosecuted in the court of law for not declaring his interest.4. Duty to prevent insolvent trading by the companyCorporate regulators have tried many means to make sure that there are no companies that can trade while insolvent by imposing higher level of liability on directors. As per s588G of the Corporations Act, civil penalties and personal liability on directors have been imposed on them if in case a company incur a debt and is declared insolvent.An example of a case where directors were liable for the companys insolvency was that of Commonwealth Bank of Australia v Friedrich. Here, maxwell Eise who was a director was fined millions of dollars for causing the company to undergo insolvency. The court argued that Maxwell Eise (Brian and Andrew in our case) had breached s588G of the Corporations Act and was liable for debts incurred by the company because his failure to prevent it.If in case directors of companies like Pandora Diamonds and Gems Pty Ltd have acted criminally and believed to be dishonest, in which may harm the company because of their self personal advantage, ASIC with the assistance of Australian Federal Police, criminal investigation can be conducted and tough procedures has to be followed when collecting evidence that will be use to determined as to whether a prosecution should go a head. And if the case is found to be more serious, it will be handled with the Commonwealth Director of Public Prosecution otherwise the lower courts will plow with these kinds of cases.All in all, directors should carefully study, understand and appreciate their duties and responsib ilities that they are subject to as company bosses. Being a director of a company is not an easy task because there are several fiduciary duties that need to be adhered to.

The impact of the Media on Teen Girls Body Image Essay Example for Free

The impact of the Media on Teen Girls Body Image EssayCosmetic makers put one over evermore sold (hope in a jar)- creams and potions that promise youth, hit, sex appeal, and notwithstanding love for the women who use them (Postrel 125). Magazines atomic number 18 filled with digitally transformed images of models with amazing bodies, flawless strip and perfectly styled hair. Television advertisers push their products using the most attractive people with the perfect bodies.Television shows much(prenominal) as October Road and movies like Men or Shoes portray images of sexy, gorgeous woman who have it all the handsome boyfriend or husband, the great job and amazing friends, while the chubby, not so attractive friend is usually there simply for comic relief. Also shows like the Janice Dickinson Modeling Agency on the Oxygen Network, is another perfect typeface of media influencing teen girls negatively. On this particular show she holds auditions for aspiring models to com e before her and audition in order to gain representation by her Modeling Agency. broada about(predicate) 97% of these young girls be harassed and ridiculed on National TV. On this show there is no such thing as rehabilitative criticism or putting it nicely. She points out every flaw, every physical feature that she feels needs to be fixed with plastic surgery or dieting. She even has gone so far as to saying I will never accept a plus size model into this agency (Rotchford 72). She has been cognise to call girls ugly and send many on occasion crying and running for the door.Her show is rated number 1 on the Oxygen Network (Rotchford 72). This human organic structure of message is sending young girls the impression that in order to be beautiful like a model and be accepted by purchase order that you have to have a flawless face and thin perfect body and at any cost necessary. The music industry has hook up withed this pattern as well. Music videos (especially of the Hip-Ho p category) are usually filled with woman wearing next to nothing, dancing erotically, and having their body parts zoomed in on by the cameras.This kind of music is extremely popular in younger generations, so when these music videos are being seen by them, then(prenominal) these scenes are going to be viewed as being cool as well. Girls will think that that is the type of look I need to have in order to be popular and cool. Teenagers are conditioned to believe that advertisements and media reflect the world. Throughout time the ideal of dish aerial has differed. During the Renaissance, a beautiful woman was more full figured and pale skinned. This reflected her identify of rank in society (Eco 212).Her size indicated she had enough to eat and her pale skin showed she did not have to work to sustain a living. During the 1920s, the image of beauty changed. Woman wore their hair bobbed, had slender figures and preferred to have smaller breasts. The 1950s brought more changes with the introduction of icons like Marilyn Monroe (Eco 306). Women wanted to be full figured, very well-endowed with platinum hair and plenty of sex appeal. As times changed and societys ideals of beauty changed, one thing remained the same, the pursuit of beauty and perfection. The most provable victim of the media stereotyping is young girls.Unrealistic images of beauty and perfection bombard these girls through television, magazines and movies. A study was performed to examine the effects of exposure of the media ideals of body image on women, and to determine if it would affect their self-esteem, body satisfaction, start eating disorder symptoms, and maybe change the level of internalization of the thin ideal. Women in the experimental group account lower self-esteem after being exposed to the thin-ideal images compared to the women who viewed neutral images (Hawkins, Granley, Richards, and Stein 44).Moreover, in some cases, feelings of body dissatisfaction and low self-esteem ma y lead adolescents desire self-improvement to increase their media consumptionresulting in a vicious circle of media exposure, internalization of sociocultural ideals, social comparison with unrealistic images, and further erosion of body satisfaction and self-esteem. Indeed one 13 year old girl in the current study commented that magazines are going to exploit the fact that teenagers are often unhappy with themselves (Clay, Vignoles and Dittmar 473). The media dictates what is considered fashionable, the popular hair styles or trends.Images of thin, leggy models in short skirts, shorts and tiny swimsuits having fun, flirting and getting attention from men visually show what is considered to be beautiful. If a girl does not fit into the mold, she may begin to have self-doubt, or body satisfaction issues. Idealized images are an influential source of pressure to meet the thin idea (Monro and Huon 89). Society is obsessed with perfection. It is evident in the number of women choosing to undergo cosmetic surgery in the sweat to attain the perfect body, nose, figure and smile.We found that girls who engage in more appearance-related discussions and imitation of others had lower appearance satisfaction (Dohnt and Tiggean 9). We found that girls who watched television shows with an appearance emphasis, such as Friends or Rage, we less satisfied with their appearance. Shows such as soap operas and music television slips that present women as thing attractive, and sometimes provocatively dressed, have also been correlated with body dissatisfaction and disordered eating in adolescents (Dohnt and Tiggean 10).Advertisement Agencies have been questioned about why they only advertise their products with a certain types of models. The response from each agency was usually almost always the same each time. Sexy sells and everyone wants to be beautiful, so we sell our products to the world using beautiful people. This shows the universe that beauty is within their grasp an d that they too can obtain it (Monroe 160). Janice Dickinson who is also in the media has stated that she says no to plus size models in her agency (Rotchford 72).We rifle in America, arent we allowed to do what we want with our birth businesses? I own this agency if I dont want fat ugly models representing it then thats my right (Rotchford 70). Miss Dickinson has a point, tho she fails to realize what her actions and her show is reflecting on to young girls. Young girls live in an appearance-focused society, where images of perfection are dust in every form of media. Girls are developing low self-esteem, eating disorders, and are striving to obtain perfection and acceptance.And the kind of media that we have is doing nothing just fueling this fire that has been burning rapidly for a long time. If young girls are not offered a more realist image of beauty, they will continue to be dissatisfied with their appearance and suffer from low self-esteem. There have been some developme nts recently to try and change this pattern. In the past few years marketers at Dove have added some new and improved enticements (The Atlantic, The Truth About Beauty 125). The new campaign declares that every woman is beautiful and ignoring imperfections indicates self-esteem.Oprah covered the story, and so did the Today show. Doves campaign, wrote Advertising Age, undermines the basic proposition of decades of beauty-care advertising by telling womenand young girlstheyre beautiful just the way they are (The Atlantic, The Truth About Beauty 125). Imagine, a beauty-care company selling their products not by enticing young girls with unrealistic images of women who achieved perfection by using their products, but by showing women and young girls they are beautiful to being with and can accentuate their beauty by using their products.Dove expanded the definition of beauty without losing the concept. This was a novel approach that seems to be catching on. Every girl deserves to feel b eautiful, smart, important and secure. Another Dove campaign focuses on young girls insecurities about their appearance. Every girl deserves to feel good about herself and see how beautiful she really is (The Atlantic, The Truth About Beauty 127). Television shows are beginning to follow in the same example as the Dove campaign.One such show is Ugly Betty, which is about a young woman from Queens who doesnt fit the standard media beauty and works for a fashion magazine where everyone is image obsessed. Instead of conforming to the standards of her co-workers, Betty maintains her integrity and shows that being a kind, smart, and caring person is what makes you truly beautiful. As time passes, people begin to see that Bettys beauty does not lie in her physical appearance but in her spirit and compassion.If images like this continue, more girls will begin to see that their own inner strength, self-assuredness, and integrity are more beautiful than the images that you see splashed cro ssways any fashion magazine or TV show. These are real qualities that any girl can obtain. Society is always going to have its own opinion of what beauty is. And this will always be perpetuated through media. If we start recognizing the impact that the media has on the self-esteem of young girls, and instead present strong, confident, and capable women achieving their goals, then young girls will being to believe that the image of beauty has a new facetheirs.

Successful Teachers Emphasize The Fun Of Learning Education Essay

Successful T to each oneers Emphasize The Fun Of Learning Education EssayAn important part of learning is having the probability to come upon mistakes, learn from them,and correct them thenext time youtry.Trainers need agreat deal ofpatience.Often it iseasierto jump in and correctthe mistake quite a than let theperson you ar training learn. on that pointfore your trainer should realise plenty of patience toensure that all individuals complete the course satisfactorily.d) Good Training SkillsThere is so much more(prenominal) to training than just reciting theory and lecturing trainees.Training sessions work much effectively when there is ahealthy interaction between the trainers and the trainees.Good trainers will toohold a professional qualification which formalises their experienceand also makes it somuch easier for any organisation to claim them .Trainers should also begood performers and make the training sessions interesting or even entertaining.In this way it becomes ev ident that the trainer is genuinely interested in memorizeing their subject.e) EmpathyA good trainer should commit a genuine interest in people , having such an interest the trainer will be fitting to get on well with them regardless of status, age, abilities or backgrounds and easily relate to their needs, aspirations and frustrations.A good trainer will have a tippy and approachable manner which make the trainees feel completely at ease andwill be particularly skilled in motivating others to achieve their goals.f) Results focussedA good trainer will not only focus on the needs of thetrainees they will all-inclusivey go out that you have your own targets to meet. motivation of trainees is one of the keyfactors of good training. A good trainer will ensure that this is achieved regardless of the individuals self-esteem or confidence.The trainerwill actively supercharge collaboration and team work between them by involving them in idea generation andproblem solving.2. Professor Some of the important qualities possessed by a professor atomic number 18a) Passion The trounce teachers are not only interested in their subject, they are passionate about it. They approach tasks with a maven of challenge rather than routine.b) Successful teachers/professors hold very high expectationsMost of the teachers expect great results from their students, and they dont accept anything less than that. Teachers have to believe that each and any student can soar past various restrictions and they will also have the confidence and work with the teachers to make it happen.c) CreativityThe best teachers are those who make their students think outside the box. The teachers make sure that the classroom experience is well understood by the students by educating them while also keeping them interested in the work at hand. They provide the students with real world applications for improving their knowledge and also taking their learning to the next action packed level.d) Patience The best teachers were those who were willing to keep explaining, knowing that eventually it would make sense to their students. They were willing to wait until a distraction calmed students down, or abandon a lesson entirely if it was evanesce material needed to be revisited. The best teachers just stuck with it, willing to do what it took, no matter how long it took.e)Teachers/Professors should be LearnersThese exemplary teachers described themselves as learners, each making it a priority to keep their teaching information current and updated. As teachers, they must continue to re-engineer the curriculum, experiment with new-fangled and different methods of delivering course content, and fiddle emerging technologies into our classrooms.f) Good teachers/professors are sensitive and versatileThe teachers who consider other peoples needs more than their own are the ones who strive to create a difference. These teachers are sensitive to the needs of students and their parents, col leagues and the communityg) Successful teachers emphasize the fun of learningThere are many different learning styles. Bad teachers only flavor a subject matter one way. They teach based on how they learn. This works for some people, but fails for others. The good teachers are ones that are able to teach to different learning styles. If students dont understand a subject, they teach it a different way. Instead of looking at abstract formulas, they explain with images what the formulas represent. This requires a thorough understand of their subject, as well as the ability to consider that subject in different ways, which not all teachers are able to do.3. Call Centre decision makera)Communication SkillsCall centres are a place of high pressure where a person will have to deal with hundreds of customers in a day. This is the reason to amend communications skills to be an expert in this field. This will help in handling different personalities, surveying skills, and vocal skills. He /She should practise on speed,volume , limpidity of voice ,voice modulation and tone of voice to attain a good communication skill.b) Listening skillsListening is one of the most valued skills companies seek in employees working in call centres .Develop listening skills through clarifying questions. Acquire feedback from your clients to develop listening skills.c) Multitasking abilityThey need to be capable of handling more responsibilities because of increased economic, regulatory and customer demands. For instance, agents need to be able to handle calls, answer emails and assist clients via Web chat. If they cant handle multiple tasks or are easily frustrated by pressure, it will be difficult for them to cope .d) Computer LiteracyIt is very necessary to be computer literate in the youthful era to be able to work with computer based software systems. Operators should be good with computer and particularly skilled in Word processing, and high-octane use of internet based operation s and tasks.e)Good Customer Care SkillsCall operators are those people who possess the necessary skills required for handling customer transaction and meeting the objective of maximising sales and productivity. An operator having an extrovert personality is an asset in the field of customer relations service centres. Agents should be able to use their unique customer relations qualities .Proper knowledge of phone etiquette is essential for excellent customer care.Q2. Design an predilection program for college students.Introducing students to college life requires presenting as full a view as possible of all that the university has to offer. Therefore, academics as well as extracurricular activities should be presented. If students do not become fondly interconnected within the first few weeks of their arrival, they are less likely to stay at that institution.1. Orientation program can start with a welcome intro , where the faculty or the dean will give the new students an insigh t into the student life at the college, gives them a strong overview of academic expectations, the different social setups on campus . This gives the advantage for students in making the transition from high school to college.2. The new students will be put in small groups , and will have an orientation leader to take them slightly . The orientation leaders can give the new students the inside scoop on college life since they too have been in the new students shoes. New students may be more open to receive information from the orientation leaders than from a lecturer .3. A campus electric circuit would be a very important aspect of orientation program. acclimating students to their new environment . For some students, going to college is their first time away from home, so orientation should give them time to become familiar with their new surroundings. Through guided campus tours orientation provides a safe avenue for new students to find their way around campus.This helps studen ts to become familiar with important locations on campus like the library , administration offices canteen, computer labs, auditoriums , hostel facilities..etc .4.Friendly interaction sessions with seniors and faculties ( over dinner or lunch )should also be arranged for the new students to feel completely at ease on campus. Illegal activities of ragging should be strictly prohibited on campus instead the new students should have a friendly association with their seniors and faculty members.These are some of the activities to be included at a good orientation program for the new college students.

GFP Practical Report

GFP Practical ReportGFP is very(prenominal) useful as a reporter protein. After its discovery in 1962 its mulish applications were put into use 30 years later by adding the coding deoxyribonucleic acid of GFP before the fetch up codon of other proteins. This each(prenominal)ows for an easily obtrusive brand nameer of the proteins presence without needing additional cofactors or causing any reproach to the organism. The spectral characteristics of GFP earth-closet be changed by making transmutations to the protein. In this investigation a Y66W mutation was made to wildtype GFP in order to produce a shorter excitation and emission wavelength. The mutation was made using QuikChange site direct mutagenesis. The protein was because ringerd into BL21(DE3) pLysS for expression. The cells were then lysed and applied to a Ni-NTA column. This divisionated the lysate in order to analyse these fractions using SDS-PAGE, fluorescence and Bradford assays. It was open up that the Y66W mutation was successfully added but cod to a nonher mutation in the stop codon additional amino acids were added to the C terminus of the protein. It was also found that purification was parti every(prenominal)y successful as GFP was eluted in the even out fraction. This is supported by the Bradford and fluorescence assays.The young fluorescent fixture protein (GFP) is a 238 amino acid protein with a molecular mass of 26,870 Da. It was first isolated from the jellyfish species Aequorea Victoria by Osamu Shimomura in 1962 (1). GFP is expressed in small photoorgans that are situated in the umbrella of the jellyfish. Douglas Prasher first realised the potential of GFP as a reporter protein (2). As proteins are smaller than the firmness power of electron microscopes, Prasher thought the GFP gene could be added into the gene for haemoglobin before the stop codon. This would allow the protein of interest to maintain all of its functions but would overhear the GFP protein at its C te rminal end. This means that detection of GFP fluorescence would also indicate the presence of haemoglobin. Furthermore, GFP does not require additional cofactors or substrates to fluoresce. This means that it works passing well as a non-invasive method of detection of protein expression. GFP is also non-toxic so it is able to be employ in vivo without causing damage or harm to the organism.Crystallisation studies (3) construct shown that GFP has a barrel structure with the chromophore buried in the centre. This chromophore is comprised of 3 amino acids (Ser 65-Tyr 66-Gly 67) that undergo a series of spontaneous cyclisation reactions to create the alert chromophore. Wild type GFP has a major excitation percentage point at 395 nm and a minor one at 475 nm with an emission peak at 509 nm. In vivo GFP is coupled to the protein aequorin which induces a blue glow when it interacts with Ca2+ ions and breaks down luciferin. This light is able to excite GFP and cause fluorescence. In vi tro this is not the case, however GFP fluorescence can be easily induced by irradiating GFP with UV light.As with all proteins, GFP can be mutated. By mutating key residues, such as residues in the chromophore, it is contingent to change the characteristics of GFPs fluorescence. The first of many mutations was the S65T mutation (4). This mutation improved the characteristics of the protein including increased photostability, fluorescence and a shift of the major excitation peak. This investigation is trading floord on the engineer of GFP to create a mutant of GFP with a shorter excitation and emission wavelength by inducing the Y66W mutation. The aims of this investigation were as follows. To carry out site directed mutagenesis of GFPuv to clone into pET28c and transform the products into XL-1 super commensurate cells. Extraction of the plasmid after incubation overnight to check the purity and niggardliness of deoxyribonucleic acid. Preparation and novelty of BL21(DE3) cells . Lyse these cells and fractionate the lysate to make pure his tagged GFP using a Ni-NTA column. Finally, detection of purified GFP by SDS-PAGE, Bradford assay and fluorescence.The workflow of the investigation can be found in figure 1 in supplement 1. A more detailed protocol can be found in the BIOC2302 semester 2 practical manual on pages 6-15 with rationale for all experiments.In site directed mutagenesis I 31 l of water was added to the PCR reaction to give a total reaction volume of 50 l.In site directed mutagenesis II a supplied culture of cells was used in the experiment rather than cells from the transformation colonies in site directed mutagenesis I.His tagged GFP was used instead of the mutant in the protein purification experiment in order for easier administration as the process is the same.Site directed mutagenesis IBefore the compressed lab work began it was first necessary to design primers for QuikChange to induce the Y66W mutation into the wild type GFP. These c an be seen as figure 2 in concomitant 2. These were created using the QuikChange primer design tool on the Agilent website.The site directed mutagenesis was carried out using the primers supplied to induce the correct mutation. The products of this were cloned into the pET28c plasmid and the XL-1 super competent cells. The cells were racing shelld as per the BIOC2302 practical manual and left to incubate overnight.Site directed mutagenesis IIUpon checking the plates in the next session it was found that no transformed colonies had gravid so a new culture was supplied. The undigested plasmid control grew approximately 50 coloniesThe culture of BL21(DE3)pLysS cells was set up and the OD600 were recorded. They can be seen in put back 1 in appendix 3. Within 50 minutes the culture had reached an OD600 of 0.483 meaning the cells were at the correct density for lysis.The cells were prepared as per the BIOC2302 practical manual and the recombinant plasmid was special(a)cted. The ming iness measured was 121.7 ng/l and the A260/A280 was 1.86 using nanodrop. Thitherfore, the ethanol precipitation was not carried out.To prepare for sequencing 4.11 l of this solution was diluted, with 5.98 l EB buffer, to the correct concentration. This was then sent to be sequenced, the results of which can be seen in appendix 4. The primer has been highlighted in green and is surrounded by a box with the mutated codon in red. A deletion also occurred in the stop codon of the mutant as highlighted by the second box with deleted bases highlighted in blue.Protein purificationThe plates were inspected in the next session. It was found that the two hundred l transformation plate grew 3 colonies and the 50 l transformation plate grew none. Transformation efficiency can be calculated for the 200 l plate as 37 transformants/g of DNA.The cells were weighed and found to be 0.539 g so 2 ml BugbusterTM used. After lysis and fractionation the SDS-PAGE samples of each fraction were prepared and loaded onto the gel.The Bradford assay was carried out while the gel ran. The BSA standards were calculated and the contents of each standard well can be seen in table 2 in appendix 3. The fractions were then diluted into their wells and the contents can be seen in table 3 in appendix 3. The plate was filled according to the map in figure 2 in appendix 5. The plate was ran and the absorbances for the BSA standards were taken from the plate readout and inputted into table 4 in appendix 5. From here a standardisation graph was set up using GraphPad Prism and can be seen as graph 1 in appendix 6. This graph shows that the data points for the standards do not fall near the line of best fit.The absorbance results from the plate readout for all of the fractions were imputed into table 5 in appendix 7. The comparison of the line from graph 1 was then used to calculate the concentration of protein in each of the fractions. All of these values were also inputted into table 5.With the Brad ford assay sail through the SDS-PAGE gel was disassembled, stained and a picture was taken. A map of the gel can be seen as figure 3 in appendix 7 and the picture of the gel can be seen as figure 4 in appendix 8. By looking at the picture it can be seen that in lanes 2, 3, 4 and 9 there are dark bands spanning the entire lane. In 5, 6 and 8 there is faint dance band across the well. In well 7 there is a distinct small band in between the 25 kDa and 37 kDa molecular markers. Lane 8 shows no bands at all.Finally the fluorescence assay was carried out as per the map of the microtiter plate in figure 5 in appendix 7. The results from the plate readout were inputted into table 5. From here a graph comparing the log of protein concentration compared to fluorescence of each fraction was plotted and can be found as graph 2 in appendix 6. This shows elution 1 with the highest fluorescence and the unbound x10 had the lowest. However, when comparing protein concentration the unbound fraction had the highest and wash 2 had none. Percentage fluorescence was also calculated and inputted into table 6 in appendix 9.The first aim of this experiment was to transform the site directed mutagenesis products into XL-1 super competent cells. The correct primers were used in order to induce the Y66W mutation into the parental DNA. However, no colonies that were meant to take up the mutated plasmid grew but the undigested control grew around 50 colonies. This means the cells did not take up the plasmid because otherwise they would have grown on the plate. This could be due to a mistake made in making the PCR reaction mixture or the DNA may have be convey damaged at some point in the experiment. Additionally, the suppliers of the XL-1 super competent cells advice to avoid large changes in temperature. This was unavoidable in this experiment and may have contributed to the cells not taking up the plasmid. In the future more care should be taken while plating and preparing the cells. in like manner preparation of any reaction mixtures should be checked very closely in order to ensure the correct reactants are added in the correct amounts.In site directed mutagenesis II the cell culture was lysed when the OD600 was 0.483. That is because E.coli cells are most likely to be made competent when they enter early log phase. This corresponds with an OD600 of 0.4-0.5.The DNA concentration extracted in this experiment was found to be 121.7 ng/l and an A260/A280 of 1.86. This means that the DNA is good quality as the desirable range for A260/A280 is 1.7-2.0 and the concentration was much higher that what was required. However, in future experiments to test for reliability multiple results should be taken. Furthermore, the data could have been confirmed by using the spectrophotometric method aboard using nanodrop.The sequencing results in appendix 4 confirmed the successful incorporation of the Y66W mutation into GFP, creating the CFP mutant. However, the second mutation at the stop codon deleted 2 bases including the first base of the stop codon. This means that when the protein is expressed the ribosome will not stop and instead will continue to add amino acids onto the C terminus of the mutant until it reaches a new stop codon. There 144 bases between the original stop codon and the next in frame stop codon meaning 48 additional amino acids will be added to the C terminus. This codon can be seen highlighted in purple below the original stop codon. These additional amino acids could affect the folding or could increase the likelihood of aggregation of the mutant protein.In the protein purification experiment the 200 l transformation plate grew 3 colonies and the 50 l transformation plate grew none. The transformation efficiency on the 200 l plate was 37 transformants/g of DNA. The reason why this is so low could be due to a number of factors such as the plating technique or the cells may not have been left to chill on ice for the optimal amount o f time. However, the negative control did not grow any colonies, confirming that all of the bacteria on the transformation plate were transformed. Again, more steps should be taken if this was to be carried out again to ensure that proper plating and prepping protocol is followed.The Bradford assay shows that in wash 2 there was no protein in the well. This means that any protein found in elutions 1 and 2 should all be His tagged GFP that bound the Ni-NTA column. This can be confirmed by the fluorescence results as the elution 1 fraction contained the majority of the total fluorescence with 44.13% of the total. However, all other fractions also produce some fluorescence. This could be due to GFP contamination in the other fractions. This could have occurred due to the resin being saturated, preventing further binding to the column. It could also be due to aggregation of the protein obscuring the His tag and preventing binding. Furthermore, the plots on the calibration graph do not f all on the line of best fit. This means that the equation of the line is not accurate and protein concentrations calculated using it are also inaccurate. Therefore, there could be more protein in each fraction than was calculated. This could account for the fluorescence in the wash and unbound fractions. The Bradford assay is quite limiting. This is due to the fact the assay only measures protein concentration rather than GFP concentration. This means that it is unsure whether the protein concentration measured in elution 1 and 2 is all GFP or it is contaminant protein. The same can be state for the other fractions, its unsure whether the protein concentration measured has been contaminated by GFP. In the future this assay should be carried out again to try and reduce contamination. The calibration graph should also be repeated until all of the data points fall on the line of best fit. Otherwise none of the calculated protein concentrations are accurate.Finally, the SDS-PAGE results shows banding in wash 1, 2 and elution 1 and 2. This suggests that there is contaminating protein in all of these fractions. Elution 1 shows a clear band at approximately the 26-27 kDa mark as it is present just above the 25 kDa marker and is well below the 37 kDa marker. This suggest the band in elution 1 is GFP as it is the grab size and is in the expected fraction.Another source of error could be due to the amount of pressure applied to the pipette. This will vary from individual to person and will affect the volume of the solution being pipetted. As such small volumes were being used and there was a lot of solutions to be pipetted it is very possible a mistake was made. This mistake would have a big aftermath on the concentration and therefore could have a big effect on the absorbance values. These errors can be avoided in future by using the appropriate pipette for the volumes being used. Further reduction in errors can come from correct technique and by doing replicates and averaging values.There could be some error in the microtiter itself. There may have been markings or scratches on the plate that werent seen at the time. This could affect how the light passed through the reader and therefore affect the absorbance values.In conclusion, the aims of this investigation were to induce the Y66W mutation into wild type GFP using QuikChange site directed mutagenesis. Furthermore, the protein was to be expressed in competent BL21(DE3) pLysS cells. Finally wildtype GFP was to be purified using a Ni-NTA column and the fractions analysed with SDS-PAGE, fluorescence and Bradford assays. The investigation successfully introduced the Y66W mutation into wildtype GFP. However, the stop codon was also mutated adding an extra 48 amino acids on the C terminal of the protein. A band indicating the presence of GFP was found at the 26.9 kDa mark in elution 1, indicating it was bound to the column and was eluted. However, all factions were contaminated with other protein .References 1. Shimomura, O., Johnson, F., and Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous. s.l. J. Cell. Comp. Physiol. 59223-39, 1962.2. Prasher, D., Eckenrode, V., Ward, W., Prendergast, F., and Cormier, M. Primary structure of the Aequorea Victoria green-fluorescent protein. s.l. Gene 111 (2)229-33, 1992.3. Ormo, M., Cubitt, A., Kallio, K., Gross, L., Tsien, R., and Remington. S. Crystal structure of the Aequorea Victoria green fluorescent protein. s.l. Science 2731392-5, 1996.4. Heim R, Cubitt AB, Tsien RY. Improved green fluroescence. s.l. Nature. 373 (6516) 663-4., 1995.