Eukaryotic Vectors for Protein Expression
Eukaryotic Vectors for Protein Expression. INTRODUCTION Expression vectors are actually the plasmids that allow the expression of the foreign DNA. Organization and expression of the eukaryotic genome are nowadays studied in vivo as it gives us the live telecast of working with eukaryotic cells. There are different eukaryotic vectors that can be brought into use for studying the expression of the eukaryotic genome. But there are some vectors which are commonly used such as yeast, animal and plant. In expression vectors it is actually the sequences more commonly called regulatory sequences that allow finished products that is protein to be obtained by means of common pathway of transcription followed by translation of the genes. Most of the drugs containing protein products manufactured by the pharmaceutical industry are made by using expression vectors only. Appropriate selection of vector for maintaining it within the host is an important part of any expression system. A promoter within an expression system can be either regulated or constituted (unregulated) one. Stable proteins are obtained if the promoter is constituted but the protein of interest that is, desired one can be obtained by making use of vector containing regulated promoter. Also most optimal combination of cell density and specific protein can be obtained in short time by making use of regulated promoter. One can easily isolate as well as purify a protein if it is exported outside the cell. Protein modifications are required for proteins produced by recombinant technology are available only in eukaryotic cells. Common host for eukaryotic expression includes yeast, insect cells and mammalian cells. Eukaryotic expression vectors are similar to prokaryotic expression vectors by many ways such as promoter, transcription, transcription and translation signal sequences. Shuttle vectors are first propagated in bacteria and then transferred to eukaryotic cells for expression as it contains prokaryotic sequences. YEAST CELLS Yeast can be grown easily on both small scale and large scale and it is also considered as a safe organism. Therefore it is used in pharmaceuticals for use in human without any approval from the government. It mostly secretes protein in a very small quantity but subsequently engineered to produce recombinant proteins which can be purified easily. VECTORS FOR EXPRESSION OF PROTEINS IN “YEAST” Different varieties of vectors are use for use with yeast and so they are classified into three main classes: Plasmid vectors like yeast shuttle vectors. Vectors that integrate into the yeast chromosome but this approach is not used mostly as it gives just the single copy of the cloned gene and is also lost in large scale production. Yeast artificial chromosomes- it is however not so convenient for use as expression vectors. Highest level of expressions can be obtained by yeast episomal plasmid but they are unstable in large cultures. Insulin, blood coagulation factors, several growth factors and several virus proteins are now produced using Saccharomyces cerevesiae. (i)Yeast Episomal Plasmids (YEps) They are first constructed by Beggs in 1978 using a naturally occurring yeast plasmid. It is 2μm long that is 6.3 kb and is found in many strain of Saccharomyces cerevesiae and has no known function. There are 50-100 copies per cell which basepairs to two unique regions each with a pair of genes transcribed from a divergent promoter. The plasmid may replicated autonomously or integrate with the chromosome. They have been extensively used in the production of either intra- extracellular heterologous proteins. They form the basis of several cloning techniques. It has transformation frequency of 103-105 transformants per μg DNA. They are actually fragment of yeast nuclear DNA and E.coli vector pMB9. There are two stages of preparation Vector pMB9 and 2 μm is cut with EcoRI and then ligated Nuclear yeast DNA digested with Pst-I is inserted in yeast hybrid. The advantages are as follows: HCN (50-100). High transformation frequency. High stability. Very useful for studying complementation. Readily recovered from yeast. The disadvantages are as follows: Recombinant vectors have been developed from this plasmid but are unstable. Novel recombinants are generated in vivo by recombination with endogenous 2μm plasmid. (ii) Yeast Integrative Plasmid (YIps) It is bacterial plasmid that can insert itself into DNA of one of the yeast chromosome. Genes integrated into yeast chromosomes are less liable to be lost by the cell as it divides than are the genes on the plasmid. Although transformation efficiency of yeast integrative plasmid is low and the copy number is one, it has proved to be useful in yeast genetics. Yeast integrative are used for placing DNA segments within yeast genome. They can be replicated and maintained in E.coli but not in yeast. It has a transformation frequency of 104 transformants per μg DNA. The advantages are as follows: Gives most stable maintenance of cloned genes. It behaves as an ordinary genetic marker. Useful for surrogate genetics of yeast like to introduce deletions, inversions etc. The disadvantages are as follows: Low transformation frequency. Chromosomal DNA needs to be cut with restriction endonuclease for recovering yeast but it doesn’t cleave original vector containing cloned gene. (iii) Yeast Replicative Plasmid (YRps) These plasmids were constructed by Struhl et al in 1979. It consists of 1.4 Kb yeast DNA fragment containing the trp1 yeast gene inserted into EcoRI site of pBR322. They remain as independent plasmids and do not integrate. Their copy number is 1-20 per cell. They carry autonomous replicating sequences (ARS) that allow them to replicate when the cell divides. These vectors have chromosomal replication origins and give rise to high frequency of transformants that is 104 μg DNA. The resulting transformants are highly unstable. The advantages are as follows: Can be easily recovered from yeast. It has high copy number. It has high transformation frequency. Very useful for complementation studies. It can be integrated into the chromosome. The disadvantages are as follows: Instability of transformants (iv) Yeast Centromere Plasmids (YCps) These are the plasmids that contain sequences around the centromere region of chromosomes and chromosomal replication region is similar to yeast replicative plasmids that is it possess autonomous replicating sequences (ARS). It has a transformation frequency of 104 transformants per μg DNA They shows three characteristics of chromosomes in yeast cells and they are as follows: They are mitotically stable in absence of selective pressure They segregate in Mendelian manner during meiosis They are found in LCN that is, 1-2/cell in the host. Advantage- they can be stably maintained. Disadvantages are as follows: It has low copy number Wild type yeast cells are force to maintain multiple Yeast centromere plasmids bearing independent selectable markers, the cells grow and cell viability is reduced indicating effect from presence of excess chromosome. (v) Yeast Artificial Chromosome (YAC) All the autonomous vectors like YEp, YIp, YCp, YRp exist in yeast as circular DNA molecules, thus none of these resemble normal yeast chromosome that have a linear structure. Also the ends of all chromosomes of yeast have telomeres as that of linear eukaryotic chromosomes. It has two telomeres one of the left and other on the right and thus prevents degradation and are required for chromosomal replication. Origin of replication that is “ori” site on the plasmid is the site where the DNA replication begins. The presence of yeast centromere helps in proper segregation of chromosome. Chromosome won’t get pulled into new cells during cell division without the absence of centromere regions. Selectable markers are also present in some yeast artificial chromosomes. They are present as single copy per cell. In actual, they are hybrids of bacterial plasmid DNA and yeast DNA. They are grown in yeast. Advantages are as follows: Have large carrying capacity It is highly stable It is very efficient because it mimics the chromosome as it has a sequence that functions as origin of DNA replication, centromeric and telomeric sequences. Large genes such as that for muscular dystrophy can be cloned in linear manner. Disadvantages are as follows: It is an inefficient system Very few clones can be prepared per μg of DNA Resolution is difficult once introduced into yeast cell Cannot be mapped by standard techniques. Eukaryotic Vectors for Protein Expression
Saudi Electronic University Amazon Corporation IT Infrastructure Report
online dissertation writing Saudi Electronic University Amazon Corporation IT Infrastructure Report.
Saudi Electronic University College of Administrative and Financial Sciences Assignment of MIS201- Management Information System MIS201 – Project 2020/2021 Submission Part 1 End of week 7 Saturday 17th of October 2020 Part 2 End of week 12 Saturday 21th of November 2020 Part 3 Instructors will manage the date and time for presentation during week 13 & 14 25 Marks Assignment Details The project involves studying the IT infrastructure of a relevant information system (IS)/ information technology (IT) used by selecting any organization of your choice locally or internationally The idea is to investigate the selected organization using the main components of IT (Hardware, software, services, data management and networking). Infrastructure Investigation, which is in a selected industry, should be carried out by using articles, websites, books and journal papers and /or interviews. In the report, you are expected to discuss: Project Report Structure: Part 1 Submission: End of week 7 Saturday 17th of October 2020Marks: 12 Cover PageThis must contain topic title, student names and Students ID, section number and course name. (you can find the cover page in the blackboard)Table of Contents (0.5 mark).Make sure the table of contents contains and corresponds to the headings in the text, figures, and tables. Executive Summary (1.5 marks).What does the assignment about, briefly explain the distinct featuresOrganizational Profile (2 marks).Brief background of the business including organization details, purpose, and organizational structure. Strategies (3 marks).Discuss different types of strategies for competitive advantages and then select and discuss the most appropriate strategies to improve the performance of the organization.Technology Involved (5 marks).How is the organization set up in terms of its IT infrastructure? Discuss the hardware, software, telecommunication, information security, networks, and other elements. Hint: You can discuss any points that you learned in this course and its related to your selected organization Hint: You can discuss any points that you learned in this course and its related to your selected organization
Saudi Electronic University Amazon Corporation IT Infrastructure Report
differences between social entrepreneurship and traditional business
One might wonder, how was social entrepreneurship come to existence? While the idea of social enterprise go back as far as to 1649 (Spreckley, 1981), the term social enterprise was first introduced by Freer Spreckley in 1978 (Wikipedia (a)). The two words, social and enterprise seem paradoxical to be put together. Entrepreneurs, generally linked to their action of making profits for themselves and the shareholders, seem unlikely to be associated with social interests. While this is not entirely true, but the typical Ebenezer Scrooge type of entrepreneurs will always be in people’s minds when describing the traits of an entrepreneur. It might just incomprehensible for some of us, how some entrepreneurs will put aside their interests in making profits for themselves for the sake of any social causes. In this essay we will discuss the similarities between the two entrepreneurship and their fundamental differences. So, what is this social entrepreneurship and social enterprise exactly? Before understanding the term social entrepreneurship, we need to firstly understand what traditional business entrepreneur itself is. An entrepreneur is an individual who owns a firm, business, or venture, and is responsible for its development (Paggu.com). In order to do so, he or she will manage the resources he had. The aim of a traditional business entrepreneur or commercial entrepreneur is to generate profits from the risks and opportunities he or she is willing to take. A business enterprise therefore would be an entity that is owned by the business entrepreneur to achieve business goals that have been set by the entrepreneur. As for the social entrepreneurs, Wikipedia define the term social entrepreneur as “someone who recognizes a social problem and usesentrepreneurial principlesto organize, create, and manage a venture to makesocial change”. Freer Spreckley, in his work Social Audit – A Management Tool for Co-operative Working (1981) describe social enterprise as “an enterprise that is owned by those who work in it and/or reside in a given locality, is governed by registered social as well as commercial aims and objectives and run co-operatively may be termed a social enterprise. Traditionally, ‘capital hires labour’ with the overriding emphasis on making a ‘profit’ over and above any benefit either to the business itself or the workforce. Contrasted to this is the social enterprise where ‘labour hires capital’ with the emphasis on personal, environmental and social benefit”. The definition of social entrepreneurship varies from a narrow definition to a wide one. Under the narrow definition, social entrepreneurship is basically the action of applying innovative means and business skills in the non-profit sector. This can be shown by a non-profit organization such as British Deaf Association for example, venturing into business to generate income. The wider definition on the other hand, refers social entrepreneurship as “innovative activity with a social objective in either the for-profit sector, or in corporate social entrepreneurship, or in the non-profit sector, or across sectors, such as hybrid structural forms which mix for-profit and non-profit approaches” (Austin, Stevenson and Wei-Skillern, 2006). In this essay, we will use the second, which is the broader definition as the definition of social entrepreneurship. There are also many types of social entrepreneurships. One might focus entirely on the social cause but other might also focus on the financial gains or profits in order to achieve its social cause. In a way, social entrepreneurship can be said as a hybrid of traditional business entrepreneurships and social objectives where social values and commercial practices are mixed. As both come from the same foundation, it is safe to assume that a social entrepreneurship will have certain similar traits as a commercial entrepreneurship would have. First, social enterprise might also focus on making profit. This for-profit social entrepreneurship will operate in the same as a commercial entrepreneurship, but instead of focusing on increasing the profits for the owner or gaining more dividends for its shareholders, this entrepreneurship will focus on gaining profits for furthering its social missions. The example of this kind of entrepreneurship would be best described by Cooperatives UK. But this might bring problem to the entrepreneurship as it would have to struggle maintaining its original missions while at the same time being competitive in the market. Another similarity would be that both social and traditional business entrepreneurships will have to mobilize their resources, be it human, financial and others in order to achieve the goal it has set. Both must consider human resources for example managers, employees and funders in the process of running the enterprise. Although there are certain differences in the way both entrepreneurships mobilize their resources, fundamentally they will consider the same things during the process. They will also have to finance the entrepreneurships. This might be through the selling of its products and services for business entrepreneurship or fundraising events for the social entrepreneurship. While both will have certain similarities between them, there are differences that make the social entrepreneurship unique from its bigger brother, commercial entrepreneurship. The first difference would be the aim, or the mission of both entrepreneurships. While traditional business entrepreneurships usually have the aim of creating profitable gains while maintaining a lower cost of production, social entrepreneurship aims “to accomplish targets that are social and or environmental as well as financial” (Wikipedia (b)) or the ‘three pillars’. For example, Co-operatives UK, a social enterprise, has the aim of “towards the creation of an increasingly successful and sustainableco‑operative economyby promoting the interests ofco‑operatives, increasing awareness and understanding ofco‑operativevalues and principles, and supporting the growth and development of new and existingco‑operatives” (Co-operatives UK). This main aim of generating profit to further the social and or environmental goals is the fundamental distinguisher between commercial and social entrepreneurship. Business entrepreneurship needs to do research for many aspects of the market before launching its product for example, the market needs and the demand of the product from the market in order to guarantee its success. For success, the market should be large and growing. Social entrepreneurship on the other hand, does not necessarily have to do the same researches as the business entrepreneurships. “A recognized social need, demand, or market failure usually guarantees a more than sufficient market size” for a social entrepreneurship (Austin, Stevenson and Wei-Skillern, 2006). But the usual problem with these social entrepreneurships is how well they use the resources they have to achieve their goals. As they have abundant of opportunities, they often miscalculated their chance and often expand without sufficient thoughts and planning been put into consideration. For example, Guide Dogs for the Blind Association (GDBA), tried to expand its operation in 1997, adding new services such as hotel and holiday programs for the blinds to its usual guide dog services. This resulted in a severe financial loss to the entrepreneurship. After scraping these services and went back to its core business in providing guide dogs to the clients, it is finally return to its better financial state. This clearly shows how improper planning nearly cost a social entrepreneurship its existence. It also shows that it is better for a social entrepreneurship to focus on what it delivered best for the cause instead of venturing into unknown areas. Despite having similarities in this area as stated before, social and commercial enterprises will also have different ways in mobilizing their resources. Commercial entrepreneurships will allocate some of their financial resources to recruit employees and able to retain them with wages and benefits while most social entrepreneurship will have difficulties in recruiting and hiring workers, thus resulting in reliance upon the volunteers. This might be due to the fact that social entrepreneurships rarely have the financial resource or incentives to recruit and retain workers. Ducks Unlimited for example, relies on the help of volunteers to raise funds. The organization has over 50,000 volunteers which held over 6,000 fundraising events throughout 2002 (Austin, Stevenson and Wei-Skillern, 2006). This clearly shows the organization’s heavy reliance on the volunteers. Social entrepreneurship will also consider different opportunities than the commercial entrepreneurship. Although both entrepreneurships will invest the scarce resources they have in any opportunities, there are several aspects that both entrepreneurships can’t overlook. Both will concern about the customers, the suppliers, the products and other economic related situations. But in commercial entrepreneurship, the focus will be on financial and economic gains while social entrepreneurship will focus on the social returns (Austin, Stevenson and Wei-Skillern, 2006). For social entrepreneurs, the social aim is obvious and clear. This will surely affects how the opportunities is looked at and assessed by the entrepreneurs. All opportunities will centre on achieving the social mission and not increasing wealth and profits. The profits gained are just merely a way to achieve the social objectives. That has been said, a traditional business entrepreneurship can and may create a change in society by tackling some social problems or even including the problems in it missions as what have been done by many large corporations nowadays, but it is not the primary purpose on which the enterprise was started. On the same side, a social entrepreneurship might also generate some profits but that is not why the entrepreneurship was started in the first place (Social Entrepreneurship). Compared to commercial entrepreneurship, social entrepreneurship also will have constraint on the type of product it offers and the market which it targeted to. Social entrepreneurship can’t change the product as it is tied to the original specified social problems which it addressed in the first place. Cancer Research UK for example, can’t abruptly change its product to support AIDS patients as it would violate its original aims and missions. The volunteers and the funds raised are for that specific cause. This ties it have create stickiness in the range of product and the targeted market. Contrary, business entrepreneurships have freedom in choosing and creating products. They can launch new line of products without having difficulties with the employees and will not have problems with getting funds. In other words, social entrepreneur might in a way just the same as any other entrepreneurs; they set up businesses and take risks in order to make profits, although some social entrepreneurs don’t stress on making profits. But that’s where the similarity ends. Asides from that, and also the consideration on how to manage enterprise, both differs greatly from each other. While in business enterprise the profit is shared among the shareholders, social enterprises use their profits towards whatever social aims they want to achieve in the first place. Therefore we can say that the primary difference between social entrepreneurship and traditional business entrepreneurship is the purpose of setting up the enterprise and how they assess their success. In a nutshell, we can see that there are many differences between the social entrepreneurship and traditional business entrepreneurship although both have similarities in certain aspects. But this can clearly be explained by the fact that the distinction between the two is not by all means a black and white distinction. No enterprise is purely social or purely economic. The difference between the two is merely how much of the traits between the two are applied to the enterprise. “Charitable activity must still reflect economic realities, while economic activity must still generate social value” (Austin, Stevenson and Wei-Skillern, 2006). But there are still differences between them. Social entrepreneurships emerged as the result of market failure where the gap is not being filled by business entrepreneurships. Social entrepreneurs are also confronted by more constraints than traditional business entrepreneurships. The limitation in resources, funding and strategy might hinder the development to achieve their missions and goals. References 1. Ashoka.org What is a Social Entrepreneur? Accessed at 5th December 2009 from http://www.ashoka.org/social_entrepreneur 2. Austin, J.; Stevenson, H.; Wei-Skillern, J. (2006) Social and Commercial Entrepreneurship: Same, Different, or Both? Baylor University 3. Co-operatives UK Accessed at 5th December 2009 from http://www.cooperatives-uk.coop/Home/about/co-operativesUk 4. Leadbeater, C. (1997) The Rise of the Social Entrepreneur, Demos 5. Paggu.com (2009) What is Entrepreneurship? Accessed at 5th December 2009 from http://www.paggu.com/entrepreneurship/what-is-entrepreneurship/ 6. Social Entrepreneurship (2006) Accessed at 5th December 2009 from http://inspired-pragmatism.blogspot.com/2006/09/social-vs-business-entrepreneurship.html 7. Spreckley, F. (1981) Social Audit – A Management Tool for Co-operative Working Beachwood College 8. Wikipedia (a) Social entrepreneurship, Accessed at 5th December 2009 from http://en.wikipedia.org/wiki/Social_entrepreneurship 9. Wikipedia (b) Accessed at 5th December 2009 from Social enterprise, http://en.wikipedia.org/wiki/Social_enterprise
Saudi Electronic University Saudi Armco Sales and Promotion Letter Paper
Saudi Electronic University Saudi Armco Sales and Promotion Letter Paper.
Consider yourself a marketing manager working in a Saudi Company. Your company has just launched a new product. The CEO asked from you to send a sales and promotion letter to the most important client of the company. This is to present the new product and persuade the managers to purchase it. Select any real product/service from Saudi market that you want to study.Write a sales and promotion letter to the most important client (preferably choose business-to-business example) of your company. Try to persuade the managers of this client-company to purchase the product by respecting the following instructions:Use some visual aids for the presentation of the product and its features (pictures, graphics, flowchart, cutaway …) Spark the imagination and curiosity of the client Announce the new product/service of your companyDescribe the different attributers and features of the new product and its functionality, specificities, added value…Express appreciation and goodwillSpecify exactly what the client should do to test or acquire the product and when Notes: The sales and promotion letter must respect the form as studied in the chapter 8 (p.229).The number of words of your letter should be from 700 to 1000.Use a maximum of 2 visual aids.
Saudi Electronic University Saudi Armco Sales and Promotion Letter Paper
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