To Build or Buy Assignment Example | Topics and Well Written Essays - 1250 words

To Build or Buy - Assignment Example The food items which are unique and entirely different from the competitor’s coffee shop would be included. There would be no sale of alcoholic beverage as the business as a theme of healthy food .There would be more of vegetable salads and fruit salads on the menu. A brand image would be created so that there would be a great impact on the consumers. A brand image would be created by giving an apt name to the coffee shop. The name would be â€Å" natura coffee shop†. This would give a touch of nature and healthy image to the business. The name of the shop is different from the Deli and can attract customer due to curiosity. It also aligns with the food sold in the coffee shop. The price of food will be reasonable as people stay away from organic food because they are expensive. The portion would be bit more and that would be a good competitive strategy to win over â€Å"Ohio Deli†. People are always gets attracted to food that deliver much more than what they pay for. As per (Miles, 2014) â€Å"Quick-service consumers, now more than ever, want to get more for their dollar, and quite often the bigger the portion the better†. The theme of the coffee shop would also be organic as we would use any synthetic decoration in the coffee shop except from the appliances. The ambience would be entirely different from â€Å"Ohio Deli†. The coffee shop would have promotional offers which would like to entertain the customer potential. The staff of the coffee shop would be young and extra polite. They would be trained to be exceptional for customer service. We also would provide food and atmosphere which is hygiene and free of pollution. A quick service will be another strategy which would keep the interest of customer in the coffee shop. Many a times customer hesitate to come back if the service is slow as the customer are employee who come for breakfast and lunch. We would introduce loyalty cards to impress the customer s

Scientific method Essay Example for Free

Scientific method Essay

Kanomycin Resistance Gene in Its Multiple Cloning Site

Kanomycin Resistance Gene in Its Multiple Cloning Site Abstract   The objective of the experiment was to engineer a pUC18 plasmid so that it contained a kanomycin resistance gene in its multiple cloning site and to transform it into cells. The kanomycin resistance gene was obtained from a pKAN plasmid. The desired plasmid was constructed by digesting pUC18 and pKAN with the same restriction enzymes, (BamHI and HindIII) and religating the products to give the engineered pUC18. The created plasmid was then transformed into E.coli strains DH5ÃŽ ±. The strains that contained the engineered plasmid were selected using two methods of selection. According to the indirect method of selection the percentage of competent cells transformed with the plasmids was 0.063% which is a low number. According to the direct method of selection on the other hand no cells were transformed. In conclusion even though some colonies with the engineered plasmids were obtained the percentage of cells transformed was very low. Also, the indirect method of selection gives better results for selection of desired strains. Introduction Bacteria can carry antibiotic resistance genes either in their chromosomes or extrachromosomally in phage or a plasmid (Hausner and de Jong 2010). B-galactosidase is an enzyme involved into the cleavage of lactose into glucose and galactose and is encoded by the lac Z gene of the lac operon. (Glick et al 2010) The lac operon is prevented from being transcribed through repression of the lac promoter. Activation of this promoter can be done by the addition of lactose or isopropyl-ÃŽ ²-D-thiogalactopyranoside (IPTG) to the medium. Lactose and IPTG simply prevent binding of the lac repressor (the product of the Lac I gene) to the promoter. (Glick et al 2010) In the following experiment plasmids pUC18 and pKAN are used to provide the genes to be transformed into bacteria. pUC18 is 2686 base pairs (bp) long and contains a bacterial origin of replication, an ampicillin resistance gene, a lacI gene, a segment of the lac Z gene encoding part of B-galactosidase (which breaks down X-gal) and a multiple cloning sequence (MCS) that is within the lac Z gene. (Glick et al 2010) The lac Z gene encoded by the plasmid is part of the B-galactosidase protein which complements a gene carried by the Escheria. coli chromosomally thus forming a functional B-galactosidase. (Glick et al 2010) If a DNA segment is cloned in the MCS then the lacZ gene will be interrupted and will not give rise to a functional protein. If that occurs then the Bacteria transformed with the plasmid will not break down5-bromo-4-chloro-3-indolyl-ÃŽ ²-D-ÃŽ ²-galactosidase ( X-gal) present in the plates. When X-gal is broken down by ÃŽ ²-galactosidase it turns blue whereas when it is n ot broken down it stays white. This color differentiation is a way to tell if there has been any DNA incorporated in the MCS of pUC18. Finally in order for the ÃŽ ²-galactosidase in pUC18 to be transcribed, IPTG has to be present in the medium so that the lac operon can be induced. (Glick et al 2010) pKAN plasmids can serve as sources for the kanomycin resistance gene. In the following experiment the kanaomycin resistance gene will be inserted in the MCS of pUC18. pKAN contains an origin of replication, a kanomycin resistance gene and multiple restriction sites. (Hausner and de Jong 2010) More importantly it contains only one BamHI and HindIII recognition sites in the whole plasmid which flank the kanomycin resistance gene. (Hausner and de Jong) This allows researchers to cut out the antibiotic resistance gene by simply using BamHI and HindIII producing only two fragments of DNA: the gene and the rest of the plasmid. Once experimenters have inserted the pKAN gene into the MCS of pUC18 and transformed the E.coli strains they need a way to select for the desired plasmid. There are two methods to select for the desired those colonies: the direct method and the indirect method. The direct selection method involves spread plating transformed strains into plates containing both the antibiotic ampicillin and kanomycin. (Hausner and de Jong 2010) Since the pUC18 plasmid confers amplicillin resistance (Glick et al 2010) and the kan gene confers kanomycin resistance (Hausner and de Jong 2010) then only the cells that contain Puc18 with the kanomycin resistance gene should be able to grow in these plates. The indirect method on the other hand is a two step selection process. In the first step the transformed strains are plated onto LB plates containing ampicillin and X-gal. (Hausner and de Jong 2010) Only the cells that have up-taken pUC18 will grow since they will be resistant to ampicillin. Furthermore ce lls that contain pUC18 with inserted DNA in the MCS will produce white colonies since they cant produce a functional ÃŽ ²-galactosidase. Cells that give rise to blue colonies will have up-taken pUC18 without any DNA inserted in their MCS since they are able to break down X-Gal. (Glick et al 2010) To select the cells with pUC18 containing the kanomycin resistance gene the white colonies are plated in plates containing kanomycin. Only the cells that have the kanomycin resistance gene in their pUC18 will grow. (Hausner and de Jong 2010) The objectives of the following experiment include the construction of a pUC18 plasmid containing the kanomycin resistance gene in the MCS, the transformation of that plasmid into the E.coli DH5ÃŽ ± cells and the selection of the cells containing the engineered plasmid. If both pUC18 and pKAN plasmids are digested with BamHI and HindIII and the digests are ligated then a plasmid which contains both kanomycin and ampicillin resistance genes should be produced; consequently cells transformed with the engineered plasmid should be resistant to both antibiotics. Materials and Methods Plasmid extraction and plasmid engineering pUC18 and pKAN plasmids were extracted from the DH5ÃŽ ± and MM294 E.coli strains respectively using a DNA isolation kit as described by (Hausner and de Jong 2010). Confirmation for proper extraction was done through agarose gel electrophoresis by running the extracted DNA in a 0.7% gel at 100V for 1 hour. The gene containing kanomycin resistance from pKAN was cloned into pUC18. The restriction digests to do the cloning were prepared as described in Table 2 in (Hausner and de Jong 2010). After plasmid digestion the kanomycin resistance gene was inserted into the multiple cloning sequence of pUC18 in a ligation reaction using the enzyme ligase and the reaction was allowed to go to completion for 24 hours at room temperature. The ligation reactions were set up according to table 3 in (Hausner and de Jong 2010) E.coli transformation and strain selection E.coli strain DH5ÃŽ ± was sub-cultured for 1 hour at 37 °C. The cells were then made competent by washing them in 10mM CaCl. Next cells were transformed with three different combinations of plasmids. The set of cells in tube 1 was transformed with uncut pUC18 DNA. The set of cells in tube 2 was transformed with cut pUC18. Cells in tube 3 were transformed with pUC18 containing the cloned pKAN resistance and finally cells in tube 4 were transformed with just water as a negative control. The transformation procedure has been described in (Hausner and de Jong 2010). Transformed cells from all tubes were spread plated onto LB+carb+X-gal plates for indirect selection. Furthermore cells from tube 3 were plated onto LB+carb+ kan plates for direct selection of cells containing pUC18 with the insert from pKAN. To determine the density of competent cells cells dilutions of , and were prepared. The two highest dilutions were plated onto LB plates. All the plates were incubated at 37 °C and they were allowed to grow for ~24 hours. After the colonies had grown on plates plate they were counted and their numbers were recorded. White and blue colonies from the LB+carb+X-gal plates were then streaked onto LB + kan plates to obtain the colonies that had the kanomycin resistance gene incorporated in the MCS. For more information on the procedure refer to Experiments in Biotechnology Laboratory Manual (Hausner and de Jong 2010) Results Extraction of plasmids from E.coli strains Figure 1 contains the image of the 0.7% agarose gel in which the isolated plasmids Puc18 and pKAN were run to check for product. As it can be seen in lane 1 a lot of Puc18 was extracted from the DH5ÃŽ ± strain. Less plasmid DNA was collected for pKAN from the MM294 strain since the band in lane 2 is of much weaker intensity. There is more than one band in lane two. The additional bands represent additional plasmids isolated from the bacteria. Calculation of Competent cell density Table 1 shows the dilutions performed on the competent cells in order to calculate their cell density. It also shows the number of colonies on the plates that were spread plated with dilution 2 and dilution 3. The results for the dilution were not used for cell density calculation since less than 30 colonies grew on the plate. Dilution was used to calculate the cell density because the number of colonies was between 30 and 300. Indirect method of selection Cells plated from tubes 2 and 3 were used to calculate the % of transformed cells. Every colony represents a single transformed cell since it can be assumed the every colony has arisen from a single cell. Furthermore for tube 3 since five plates were spread plated the percentage of the transformed cells was obtained by using the average amount of colonies for all five plates. Calculation the percentage of transformed cells in tube 2: %of transformed cells= x 100 =0.0045% of cells transformed Calculation of transformed cells in tube 3 Average for blue colonies: = 58.6 ≈ 59 blue colonies Average for white colonies = 11.4 ≈ 11colonies Total number of colonies = 59 blue colonies + 11 blue colonies = 70 colonies in total Both blue and white colonies from tube 3 represent transformed cells since they both up-took plasmid DNA whether it was just pUC18 or pUC18+kanomycin resistance gene. Therefore since every colony came from a single cell there were 70 cells in total that were transformed from 100 µl of media spread plated in each plate. % of transformed cells in tube 3: %of transformed cells= x 100 =0.063% of cells transformed Direct selection of clones containing the kanomycin gene: No colonies grew on LB + carb + kan plates. That means that there were no cells that were transformed with the engineered plasmid. Furthermore an accurate number for % of transformed cell could not have been calculated even if cells had grown in these plates. That is because this selection method takes into account only the cells that were trasformend with pUC18 which contained the kanomycin resistance gene and not the cells that were transformed with only pUC18. Discussion Isolation of plasmids from cells The optimal results for the gel would have been to see one strong band at ~2.7 kb representing pUC18 and one strong band at 4.2 kb which represents pKAN. For the pKAN lane there is more than one band seen. Those bands represent different sized plasmids that were also isolated from the cell. Since there was no DNA ladder on the gel it cannot be concluded what plasmid the lanes represent but the only thing that can be concluded is that there was plasmid DNA isolated from both the DH5ÃŽ ± and the MM294 strains which most likely was pUC18 and pKAN. In order to conclude whether pUC18 and pKAN plasmids were isolated from the bacteria the students should be provided next time with a DNA ladder in order to determine the sizes of the lanes. Indirect selection method The cells from tube 1 were transformed with un-digested pUC18. The cells from this tube represented a positive control for transformation. The colonies in the plates were all blue and they were too many to count. The reason for the high number of colonies was that these cells were transformed with undigested plasmids which are all stable and all allow bacteria to carry information extrachromosomally, making the transformation percentage of competent cells very high. All the cells from tube 1 produce blue colonies. That is because they all had a functional B-galactisidase since no genes were cloned into the multiple cloning site located within the lacZ gene. The cells from tube 2 were transformed with digested pUC18 plasmid. The cells from this tube represented a negative control for kanomycin resistance gene cloning. Tube 2 gave rise to very few colonies in comparison to tube 1 because the cells in tube 2 were transformed with unstable DNA. pUC18 had been previously digested with HinDIII and BamHI and a lot of plasmid did not re-ligate and for that reason the DNA was unstable. Since the DNA was unstable it was not able to maintain the ampicillin resistance gene in bacteria and consequently the strains were not able to grow in carbonicillin plates. As a result the number of percent transformed cells was as low as 0.0045%. The cells from tube 4 were transformed with sterile water i.e no DNA. These cells represented the negative control for transformation. Because no DNA was inserted in them none of the cells contained the ampicillin resistance gene and as expected none grew in the plates containing carbomicillin. The cells from tube 3 were transformed using pUC18 that contained insertion on the MCS as well as pUC18 that didnt. All five plates that were spread plated with E.coli from tube 3 contained blue colonies as well as white ones. The reason for the color difference is that the blue colonies contained a functional ÃŽ ²-galactosidase whereas the white ones didnt. The functional ÃŽ ²-galactosidase in the blue colonies was due to the fact that no DNA was inserted in the MCS to interrupt the lacZ gene. The white colonies on the other hand did not contain a functional ÃŽ ²-galactosidase since they had a DNA insertion in their multiple cloning site, which interrupted the lacZ gene. Consequently they could not break down X-gal. However just because they had a DNA insertion in their MCS it did not mean that they contained the kanomycin resistance gene. They might have contained the rest of the pKAN plasmid. As a result the white colonies needed to be streaked into plates that selected for kanomy cin resistance. If the cells then grew on LB + Kan plates and they also originated from white colonies on LB + Carb + X-gal plates then they contained a Puc19 plasmid with a kanomycin resistance gene inserted in the MCS. The percentage of transformed cells was also not very high: 0.063%. A way to improve this would be to maybe increase the molarity of the CaCl solution to make the cells more competent. Direct selection method According to the direct method of selection there were no cells that were transformed. This is contradictory to the results obtained from the indirect method of selection. This error could have been produced because of either improper spread plating of plates or because of improper transformation procedure. Also the conditions in the LB + carb + kan plates could have been too harsh (two antibiotics) for the bacteria to pick up growth even if they were resistant to both antibiotics. In following experiments it is better to use the indirect selection method since it seems more successful in selecting desired strains. Comparison of direct VS indirect selection methods The direct and indirect selection methods have both advantages as well as disadvantages. The main disadvantage of indirect selection is that it takes longer since it contains two steps and each step takes at least a day for completion. The main advantage is that if done correctly, the indirect selection methods gives very accurate selection for the desired cells. The reason for that is that first it selects for colonies that just have an insertion in the MCS and this tells the researcher that some type of cloning has occurred in plasmids. The second step then selects for the colonies that contain pUC18 with the kanomycin resistance gene inserted in the MCS. Thus the criterion of indirect selection is that cells have both pUC18 with an inserted DNA in MCS and also have kanomycin resistance. The colonies that grow in the second step fulfill both the criteria. The main advantage of the direct method is that it takes a shorter time to complete and it also uses up less equipment which can also save researchers some money. The main disadvantage with this selection is that it has a higher chance of giving false positives. Direct selection does not select for strains that have DNA inserted in the MCS of Puc18 but only selects for strains that have ampicillin and kanomycin resistance. Therefore the strains that grow in LB + carb + kan plates might have both pUC18 and pKAN plasmids but not the kanomycin resistance gene inserted in the pUC18 MCS. Those strains would still be able to grow since they still have both ampicillin and kanomycin resistance. However the genes would on different plasmids and not on the engineered one. Therefore even though the indirect selection method is longer it is more accurate in selecting the desired strains for this experiment. In conclusion, according to the indirect selection the desired plasmid was engineered by digesting both pUC18 and pKAN with HindIII and BamHI. Also when selecting for cells transformed with pUC18 it is better to employ the indirect method of selection because it gives more accurate results. Question 1: Although both lanes contain plasmid DNA, why doesnt the DNA appear to be in the same location in both lanes? The DNA does not appear in the same location in both lanes because pUC18 and pKAN are of different sizes. pUC18 is 2686 base pais long whereas pKAN is 4194 base pairs long. (Hausner and de Jong 2010) Because pUC18 is of smaller size it will travel farther from the wells than pKAN. Question 2: How would you verify that the transformed cells actually contain the carb/kan plasmid that was used for transformation? One accurate way would be to isolate the plasmid DNA from the transformad cells and run it on an agarose gel. If the kanomycin resistance gene was inserted into pUC18 then on the gel one will be able to see a band of the size 4548 base pairs which is different from both the pUC18 and the pKAN plasmids. The size of the created plasmid was calculated the following way by obtaining the information from (Hausner and de Jong 2010): To find the size of kanomycin resistance gene inserted in pUC18, the number of base pairs from the origin or replication of HindIII was subtracted to the number of base pairs from the origin of replication of BamHI. This was done because pKAN was digested with HindIII and BamHI to obtain the kanomycin resistance gene: 2095 233 = 1862 base pairs The size of the insert was then added to the size of Puc18: 2686 + 1862 = 4548 base pairs

Bush Imposes Gag Rule :: Argumentative Persuasive Topics

Bush Imposes Gag Rule On January 25, 2001, on his first business day in office (and the 28th anniversary of Roe v. Wade, the landmark U.S. Supreme Court decision establishing a woman's right to an abortion), President George W. Bush stupidly re-imposed the Global Gag Rule on the U.S. Agency for International Development (USAID) population program. This policy restricts foreign non-governmental organizations (NGOs) that receive USAID family planning funds from using their own, non-U.S. funds to provide legal abortion services, lobby their own governments for abortion law reform, or even provide accurate medical counseling or referrals regarding abortion. See what damage he is doing! The 1973 Helms Amendment is a legislative provision that already restricts U.S. funds from being used for these activities, but Bush had to get involved for political purposes. About 2 million women die every year from unsafe abortions, a statistic that could be virtually eliminated by the provision of appropriate health information and services and law reform efforts. Despite this, President Bush's Executive Memorandum directs USAID "to reinstate in full all of the requirements of the Mexico City Policy in effect on January 19, 1993." According to this policy, foreign organizations--often the only health care providers in remote, rural areas--are prohibited from using their own, non-U.S. funds for: * providing legal abortions even, can you believe, where a woman's physical or mental health is endangered (the only exceptions are in cases of rape, incest, or where the woman's life is endangered); * providing advice and information regarding the availability and benefits of abortion and from providing referrals to another health clinic; * lobbying their own governments to legalize abortion, to maintain current law and oppose restrictions, or to decriminalize abortion; and * conducting public education campaigns regarding abortion. In addition, even the provision of services that are "permitted"1 on paper, such as life-saving abortions and post-abortion care, are often curtailed because NGOs fear jeopardizing their funding through any association with abortion - what cowardice!! Providers may even be reluctant to dispense emergency contraception--which acts to prevent pregnancy and is not an abortifacient (despite the lies you may hear from the antichoice groups and the Catholic Church --because of the Global Gag Rule.

P. S. I Love You Analysis

The movie I chose was P. S. I Love You. To begin with, I chose this movie because I have not seen it yet, but I have heard from my friends that it was a movie that I cannot miss out on. Reading the captions of the movies, it seemed very interesting and as soon as I was done with the movie, I gladly found it interesting. P. S. I love you had a unique plot compared to other love movies, after reading the synopsis I could automatically relate this movie with Chapter one on â€Å"Myth of the right one†. P. S. I Love You introduce its self with the daily lives of Holly Kennedy (the main character) and her husband Gerry Kennedy, a happily married couple going through their up and down stages of their marriage. Until Gerry dies of a brain Tumor, it takes Holly about a year to get over, but the movie narrates her life throughout the year. Holly and Gerry were married for 10 years and she got married at the age of 19, so we can say she married an age where she believed that Gerry was the right one and there would be no other like him. After the death of Gerry, Gerry understood that it would be hard for Holly to get over him so on his last few days before his death he writes several letters instructing her what to do as a last request. Holly received her first letter shortly after Gerry’s funeral on her 30th birthday. Gerry along with Holly’s friends are determined to to make Holly move on from the widow stage and be herself. Throughout the letters (which he had arranged for after his death) Gerry ends them by saying P. S. I Love you. The letter are supposed to encouraged Holly to move on by going out more and spending more time with her friends (Denise and Sharon), but her friends are afraid that the letters are keeping Holly tied up. In one of the final letters, Gerry arranged Holly, Denise, and Sharon, to his home town in Ireland. Once in Ireland, the girls are having fun at a local pub Gerry had recommended them to. At the pub, Holly meets a singer who strongly reminds her of her dead husband Gerry and ends up liking him, but by coincidence he happened to be one of Gerry’s best friends. Throughout the Ireland trip Holly finds out that Denise was having a baby and Sharon is getting married, so it caused Holly to emotionally relapse into herself and get depressed once again. Weeks later in New York Holly believing she was done with her letters until she received her final one from her mom when she was crying to her about how hard it was to go on without the love of her life. But in the last letter Gerry says his final good bye and tells Holly to move on and be herself and to think about what she was before she met Gerry (an Art Major). Eventually Holly finds out she has the talent to design women shoes. As Holly starts her own line of shoes we see that she gained a new confidence and it allows her to finally accept herself for what she has and for her friends to finally experience happiness. So the movie ends by Holly taking her mother to Ireland and as the film ends it shows the audience with a scene where she finally abandoned the fear of falling in love again and has opened up her life to the journey that awaits her. The Character I’m choosing in the movie to explain the problem that the character is facing is Holly. At the beginning of the Movie before Gerry died, Holly believed that he was â€Å"the one†, but then after his death, she goes on a journey that Gerry had left planned for her. Throughout the Journey Holly meets other men and fights her fear of falling in love again, believing that only Gerry was â€Å"the one†. At the end of the movie Holly learns how to accept Gerry’s death and meets a new man. If I were to choose a chapter from the book â€Å"A Daring Promise†, it would have to be the first chapter. As I was explaining throughout the whole essay, Holly is in the category of â€Å"Myth of the Right One†. She believes that there’s only one Gerry out there even after his death, until finally after a year of going through Gerry’s planned letters and other events, she finds a new man, that changed her image of â€Å"the right one†. So is there really only one right one for us? The book tells us that there isn’t such thing as one love, it’s just a myth. Based on the text, I can state that â€Å"One Love† is really a myth. There is more than just â€Å"One Love†. To obtain â€Å"One Love† both partners must always be communicating and come to agreements. In my opinion many people at first believe in only â€Å"One Love† at first because they have not had many relationships. We must come to our senses that nobody is perfect and we have to accept both the good and bad of a person. If you and your partner are willing to make sacrifices, then together you guys can make changes and have the same love for each other and being more united. If there is something I have learned from this assignment, it would be about marriage. I have never experienced a dead wife but I would assume that anyone who loves their partner would go have to go through what Holly did. Throughout the movie I also learned that getting married and losing a husband or getting divorced can affect everything around your life. When Holly was with Gerry they would always argue about when to have kids and when to move out of the apartment, but when Holly was single she lost her main balance of her life and things were pretty hectic in the beginning, until she finally had the support of her family and friends. Marriage is a great responsibility. I have also learned in class that you cannot change a person unless that person is willing to change.

The Rise of Nationalist Movements essays

The Rise of Nationalist Movements essays In the rise of nationalist movements and modern nation-states in the 20th century, women are actively participating in the movement for liberation. Throughout the world, much of the liberation of former European colonies and creation of new states stemmed from the active role women took in the struggle for independence. The documents included in this question relate to how the role of women has changed and how it has stayed the same. In the documents, women of different nations individually speak out on issues such as equality, social responsibility, and the traditional cultural views on women. One group of documents - #1, #4 – relates the view that the participation of women in their country's liberation enables them to achieve equality in their standing with men. According to "An Indian Freedom Fighter Recalls Her Life," (#1) Manmohini Zutshi Saghal recalls that the satyagraha, the nonviolent resistance approach developed by Gandhi, included women and thus allowed them to participate in processions and Congressional meetings. Teodora Ignacia Gomes (#4) of the African Party for Independence of Guinea and Cape Verde in 1974 makes clear that women need to participate in the national struggle for independence in order to achieve their self liberation. Manmohini, a participant in the Indian struggle for independence, portrays the role of Indian women as restricted before the movement of 1930 – 1932. The success of the satyagraha lay in the fact that Indian women were allowed to participate in the liberation movement of India. The traditional cultural gender role of women was to stay home, except to visit relatives or attend religious festivals. For women, the opportunity to "leave their homes and walk in a procession was a big step forward." Teodora Ignacia Gomes argues the point that women need to participate in constructing a society free of exploitation so that they are liberated from the bonds traditional ge...