Gene chlR in Streptomyces Venezuelae

Gene chlR in Streptomyces Venezuelae PCR amplification and overexpression of the positive regulatory gene chlR in Streptomyces venezuelae J. L. CLAYTON BROWN Introduction The polymerase chain reaction (PCR) is a technique used in the amplification of DNA which utilises thermal stable polymerase, Thermus aquaticus (Taq) and primers which aid in the annealing of the chosen DNA strand, producing numerous replications through a cycle of appropriate temperature changes (Lorenz, 2012).   Developed in 1983 by Dr. Kary Banks Mullis, PCRs ability to quantitate transcription levels of specific genes has revolutionised research and the understanding of gene function (Bustin, 2000) in its many applications, including the ability to: detect DNA polymorphs and point mutations (Orita et al., 1989), amplify specific genes for the construction of overexpression vectors (Liang et al., 2015), and recognising bacterial (Hill, 1996) and viral (Holodniy, 1994) pathogens. Advances within PCR have only broadened the spectrum of its implementations, with new techniques such as Quantative PCR, and Inverse PCR yielding new insights into once misunderstood areas of molecular b iology (Jain and Varadarajan, 2013). ChlR is a cluster-associated transcriptional activator consisting of 987 base pairs within the putative CHL biosynthetic operon, predicated to encode the only positive regulator responsible for the initiation of production of chloramphenicol (CHL) (Fernà ¡ndez-Martà ­nez, et al., 2014); with the usance of PCRs cloning capacity, it is predicted that the introduction of a plasmid capable of overexpression of the chlR gene will result in amplified activity of the CHL biosynthetic gene cluster. Method DNA amplification by means of PCR often requires a high fidelity taq polymerase within the PCR mixture to minimise mutations (McInerney et al., 2014). The chlR DNA fragment was inserted into the vector pIJ10257 [1] prior to PCR. The final reaction mixture consisted of the following: 10ÃŽ ¼l 5X Colorless GoTaq ® Reaction Buffer, 2ÃŽ ¼l PCR Nucleotide Mix (10mM each dNTP), 5ÃŽ ¼l Apra_BamHI_F primer, 5ÃŽ ¼l Apra_BamHI_R primer, 2ÃŽ ¼l purified chlR chromosomal DNA, 5ÃŽ ¼l Dimethyl sulfoxide (D MSO), 20ÃŽ ¼l Nuclease-Free water, and 1ÃŽ ¼l GoTaq ® G2 DNA Polymerase, equating to a total reaction volume of 50ÃŽ ¼l within a sterile, nuclease-free PCR tube, labelled as group 2. The appropriate annealing temperature was set accordingly with the melting temperature (Tm) of the hybridising portion of the primer. The extension temperature was calculated upon the approximation of 1 minute per every 1kb of required amplified DNA. The resulting thermal cycle was applied as such: an initial cycle at 95 °C for 5 min (denaturation), 95 °C for 30 secs (secondary denaturation), 56 °C for 30 secs (annealing), 72 °C for 90 secs (extension), repeated for 30 cycles from secondary denaturation. The final extension temperature was 72 °C for a period of 5 mins (holding temperature 10 °C). Gel electrophoresis (GE) was preformed using a 0.8% polysaccharide agarose gel within a Tris/Borate/EDTA (TBE) buffer and inserted into the 2nd column. Results Gels were removed from the gel box and inspected underneath a UV light. No band of DNA was visible within column 2; the molecular weight ladder and chromosomal DNA within columns 3, 13, and 16 from other accompanying PCR (run simultaneously under the same conditions previously described) were observable. When compared to the molecular ladder, successful DNA fragments indicated a base pair (bp) length of approximately 1000bp, in correlation with the 987bp of the chlR regulatory sequence, an indication that the inserted DNA is present. Fig.1   0.08% agarose gel exhibiting the DNA ladder and S. veneuzlae chromosomal DNA within column 3, 13, and 16. Column 3 shows an excessive quanity of DNA, an indication overproportionate amount of template DNA were added during procedure. The absence of DNA within column 2 (indiciated in red) evinces the failed PCR described in this paper. Discussion   Ã‚   The absence of DNA within column 2 demonstrated the failure to obtain a PCR product. As each component was correctly incorporated, other aspects must be adjusted to result in an adequate amount of DNA cloning. There are several alterations implementable to increase the likelihood of success within the reaction, firstly being the redesign of appropriate primers as the most crucial component for successful amplification of the reaction (Dieffenbach et al., 1993); analysis based software for enhancing the specificity of the primers without compromising their sensitivity can be implemented, with programs such as Primer3 and QuantPrime offering the possibilities of designing internal oligonucleotides alongside primer pairs, and the optimisation of these primer pair designs enabling specificity evaluation, respectively (Noguera et al., 2014). If the primers present correctly, changes to the temperature cycle should next be ensured. A decrease in the annealing temperature has previously shown to reduce the risk of unspecific binding and preferential amplification (Sipos et al., 2007). A final modification to the protocol is to adjust the number of PCR cycles, as this change can influence aspects of the reaction; a low PCR cycle number may provide accurate estimation of bacterial richness and a decrease of PCR errors (Ahn et al., 2012), whilst an increase in cycles can improve fluorescent intensity of some dyes (SYBR ® Green I) (Ramakers et al., 2003). Electroporation is a common method of transformation concerning plasmids, involving a brief high-voltage pulse which renders the membrane pores to transiently open and allow the subsequent uptake of DNA into the host cell (Pigac and Schrempf, 1995); an associated example is an electrotransformed Escherichia coli bacteriumwith a cloned, overexpressed chlR gene. In order to clarify correct insertion and amplification of the correct sequence, the DNA must be sequenced. The most common method of DNA sequencing for cloned PCR products is the Sanger sequence, which technique lies in the use of chain-terminating nucleotides (Sanger, et al., 1977). Once clarified, the replicative vector can then be transferred to S. venezuelae via coagulation from the E. coli, transferrable due to the origin of transfer (oriT) within the vector (Mazodier et al., 1989). It Is expected that an overexpression of the chlR gene would result in elevated levels of the encoded transcription factor protein, initiating increased transcription of the CHL biosynthetic cluster and producing a higher chloramphenicol yield; this would be observable through analysation via High Performance Liquid Chromatography, a sensitive method appropriate for gene expression analysis (Sivakumaran, et al., 2003).Recent research [2] has strongly indicated that the constitutive expression of chlR effects the overall expression of the speculated, proceeding genes within the cluster, confirming chlRs role as a transcriptional activator (Fernà ¡ndez-Martà ­nez et al., 2014).

An It Proposal for Network Management of the College of Computing and Information Sciences(Makerere University, Uganda)

AN IT PROPOSAL FOR NETWORK MANAGEMENT OF THE COLLEGE OF COMPUTING AND INFORMATION SCIENCES(MAKERERE UNIVERSITY,UGANDA) Introduction The College of Computing and Information Sciences (COCIS) Makerere University in Uganda was established on 13th December 2010. It is made up of the School of Computing and Informatics Technology (CIT) and the East African School of Library and Information Science (EASLIS). COCIS is one of the largest computing and ICT training, information science, research and consultancy colleges in Africa.It cuts across as a world class College and has maintained its place as an icon of excellence on the continent. It is committed to delivering excellent services in the area of Computing, Library, Records and Information Sciences and attracts both local and international students. COCIS boasts of the state of the art infrastructure including lecture theaters, giant computer laboratories, specialized computer laboratories and a college library.The established academic staff strength of the College is about 300 teaching staff including 30 visiting and local professors. Over twenty of these staff members hold doctorate, with many others in advanced stages of completing their PhD studies. This merger has seen a pool of resources and facilities like computer laboratories, specialized laboratories, book libraries, journals, cameras, and personnel both administrative and academic staff.To maximize productivity and efficiency, the college of computing and informatics technology(CoCIS) routinely relies on the network to support its operations ranging from E-learning activities on the E-Learning environment(Muele) to support services offered to students and staff ,services like access to wireless internet, access to the internet through the Domain(CoCIS domain) The network topology used by the college is an extended star LAN that is used with network devices that filter frames or packets, like switches, and routers.This topology significantly reduces the traffic on the wires by sending packets only to the wires of the destination. This network is managed by only four people or staff. This network is also being managed using tools like Nagios and solar winds for monitoring devices. Problems identified affecting the network Insufficient resources Under this there is limited human resources which include support staff and network administrators . there are only four network administrators for the whole college of which some of the are incompetent or not always available.Limited number of Computers, most of the computers in the labs at the college are dwindling in numbers due to technical problems, a few switches and insufficient number of Ethernet cables of which some don’t work, one of the servers at Block A has issues and It has really disrupted the network mainly during the access to the domain by the students. Low bandwidth The bandwidth that is provided for the college by the service providers (UTL) is not enough to cater f or the students and staff that are the end users of the network.Delayed Service delivery and network instability There is slow network connectivity in the labs and other areas of work in the college for example these days lecturers rarely access Muele(Makerere E-Learning environment) to upload Notes for students due to this problem. Network intrusion / unauthorized usage There is increased network access by unauthorized users on the Wireless network that increases the network traffic and hence slowing down of the network which deprives the rightful users who are the students of the college from fully utilizing this resource.Security issues The College faces security problems which include theft of network devices like cables, mouse, keyboards et cetera. Also to note that much as there are measures for security like cameras in some labs and strategic areas of the buildings, security men for the two blocks; they are not enough Proposed Solutions to the above mentioned Problems at CoCI S Network management being a complex and broad component in networking, it can be achieved through a number of networking frameworks.So we recommend that if this college can use the following approaches, its network management can be eased and improved tremendously. Fault management The college network administrator should be able to identify all the network faults like no connection or slow connection , locate where the fault is ,restore the service ,identify the root cause of the fault and then find a resolution for the problem. This can be done proactively or reactively. Hence the network downtime is minimized highly. Configuration managementThe network administrators of the college should be able to capture network and system configuration information of all network elements. (local, remote, automated and manual) ,Map the network topology, Set up configuration parameters in management agents ,track and document what changes are made to the network, where they are made and why th ey are made , Identify where upgrades need to be made so as to manage the network efficiently as they curb problems of delayed Service delivery and network instability (physical and logical configuration).Accounting management Since there are limited resources like computers, human resources that is to say administrators of the network ,distribution of these resources can be done optimally and fairly on the college so as the network is managed well . This makes the most effective use of the systems available, minimizing the cost of operation. Security Management Security measures should be adopted so that the network is protected against unauthorized users, and physical or electronic sabotage hence mitigating issues of network intrusion.The security systems should also allow network administrators to control what each authorized student or staff can and cannot do with the system. Other security measure to adopt can be: -Physical security This can be done by providing enough security cameras in all the labs and other areas in the buildings of Block A and Block B, locking the computer labs and mounting servers on cabinets or racks that have locks.Proposal to the top administrators to purchase enough Bandwidth to suit the growing numbers of users at the college. If there’s enough bandwidth provided, it will cater for the slow connections on the network as earlier indicated. Purchase of needed network equipments like extended servers, routers, better tools for monitoring the network should also be done to stabilize the network infrastructure. CONCLUSIONThe college of computing and information sciences needs to aim at putting the above mentioned measures into consideration and practice where possible so as to enable full utilization of resources which minimizes redundant resources, gives higher chance to students and staff to access resources on the network without inconveniences, quick service delivery for the students and staff will be guaranteed reducing on the downtime experienced on a slower network and also curbing time delays in all the network operations.References CIS. (n. d) Retrieved from Cis. mak. ac. ug:http://cis. mak. ac. ug/about-cocis/history-a-facts/the-college. html Makerere E-learning Environment (n. d) Retrieved from Muele: http://muele. mak. ac. ug/

Economic freedom Essay

Does economic freedom help explain why the standard of living improves in some countries and declines in others? First of all we have to understand, what economic freedom is? Economic freedom according to William W. Beach and Tim Kane, PhD. â€Å" Economic freedom is that part of freedom that is concerned with the material autonomy of the individual in relation to the state and other organized groups. An individual is economically free who can fully control his or her labor and property.† (Taken from 2007 Index of economic freedom chapter no.3 by William W. Beach and Tim Kane, PhD). Or in simple words we can also say that everybody in his or her own country is said to be economically free, if their respective government protects their rights in every possible way and without any constraint. i.e. if an individual wants to learn anything, to do any kind of business or wants a job, then, he or she has a right to do that, while it is the responsibility of the government to make it convenient and possible for them. The first study of economic freedom was published in the year 1995, and it was improved over the years. There are about ten different types of economic freedoms, which are known as index of economic freedoms. The main purpose of defining these freedoms is to rank the countries in order to pave the way for foreign investment. Following is the list of ten economic freedoms: (taken from 2007 Index of economic freedom from chapter no.3 by William W Beach and Tim Kane, PhD). 1) Business freedom: To check the status of starting, operating and closing any business in how much time. 2) Trade freedom: To check the barriers and constraints in tariffs, import and export in the country. 3) Monetary freedom: To check the measure of price stability and how governments are controlling the prices, inflation etc. 4) Freedom from government: To check either state is providing public goods with minimum expenditure or not. 5) Fiscal freedom: To check the burden on the government with respect to revenue side. 6) Property rights: To check whether the public have keeping the private property rights, defined and secured by the laws, and are clearly defined by the government. 7) Investment freedom: To check whether the government deserves to have the foreign investment or not. 8) Financial freedom: To check the operation of financial institutions i.e. banking systems; are they out of government control or not. 9) Freedom from corruption: To check the status of corruption in the society, i.e. in the business system, judiciary, and administrative system in the country. 10) Labor freedom: To check the status of growth of labor and business either they are working without government interruption or not. (Taken from 2007 index of economic freedom from chapter no.3 by William W Beach and Tim Kane, PhD). Each one of the ten freedoms is measured on a 0-100 percent scale, 100 as the maximum freedom while 0 is the least or minimum freedom. The study of economic freedom was conducted on about 157 countries of the world. To show how it works, I choose two countries that are North Korea and South Korea. North Korea has a communist form of government and is ranked at no.157 in the index of 157 countries, its economy is 3% free. It has exports of $1.3 billion, it exports: minerals, metallurgical products, manufacturing (including armaments), textiles, fishery products etc. and imports of   $2.3 billion, it imports: petroleum, coking coal, machinery and equipment, textile grains etc. In communist form of government every asset in the country belongs to the state, therefore there is no business freedom i.e. 0 % (in 0-100 % scale) in N. Korea. Also government controls all the imports and exports so trade freedom i.e. 0 % is also nil. No data on income or corporate taxes is available so fiscal freedom i.e. 0 % is also zeroed. Also government owns all the property including businesses therefore freedom from government i.e. 0% is also nil. No publicly record of inflation data therefore monetary freedom i.e. 0% is also zero. Also no private financial sector, therefore financial freedom i.e. 0% is also nil. Wages and incentives are also controlled by government therefore labor freedom i.e. 0% is also nil, also corruption is heavily present in the country so corruption freedom i.e. 10% is also negligible, but there is some investment freedom i.e. 10%, after the opening of foreign investment zone in the far remote area of rajin sonberg where basic facilities are also not provided till date. The government does not cover property rights i.e. 10%, so they are also negligible.   (Taken from 2007 index of economic freedom from chapter no.5). While South Korea has democratic form of government and it is ranked at no.36 out of 157 in the index of economic freedom, and its economy is 68.6% free. S. Korea has export of $299.2 billion, it exports: semi conductors, wireless telecommunication, equipment, motor vehicles, computers, steel, ships and petrochemicals etc. It has imports of $269.8 billion, it imports: machinery, electronics, electronic equipment, oil, steel, transport, organic chemicals and plastics etc. As South Korea has democratic form of government, in that form, the government tries every possible effort to please its citizens, therefore starting, operating and closing the business in that country is relatively easy, there is a lot of business freedom i.e. 83.1% present in the country. S. Korea as compared to N. Korea has good trade freedom i.e. 64.2% present but there are some restrictions in its some activities like import, non-transparent regulations and standards etc. S. Korea has high income tax rate of about 38.5% and relatively low corporate tax of about 27.5%, so we can say that it has a good fiscal freedom i.e. 0-100 bracket it has about 81% free. After the foreign investment promotion act of Nov 1998 the government opens the doors to foreign investment in almost every sector except media, electric power and some agricultural sectors, and also residents and non residents can now holds foreign exchange accounts, so there is a lot of investment freedom i.e. about 70% free. The private property right i.e. 70% is secure by the government; corruption i.e. 50% is present at some extent in the country. The labor i.e. 57.7% market is working under restrictive employment regulations due to which employment and production growth rate is very low. Government interference in private sector is negligible, so freedom from government is 81.5%. The government regulates the prices in some sectors like agriculture, telecommunication and other utilities, which monetary freedom 79%, the government in retained some ownerships, second largest domestic bank is under them, which makes the financial freedom about 50% free. (Taken from 2007 index of economic freedom from chapter no.5).   In short economic freedom in South Korea is much superficial than North Korea, and it is mainly due to democratic and communism forms of governments, democratic people are enjoying the fruits of freedom while others do not.

Whats Marital Labor and as a Business Owner How Will it Affect my Divorce - Free Essay Example

Sample details Pages: 2 Words: 531 Downloads: 6 Date added: 2019/06/24 Category Psychology Essay Level High school Tags: Divorce Essay Did you like this example? Like the residents of Florida, all Florida divorces are distinctive and individual in their own way. When contemplating divorce, sometimes it is informative to find divorces that somewhat mirror your situation so that you can allow the rulings within that case to guide your own preparation. Your Orlando Divorce Attorney will guide you through previous case law, as well as rules and regulations in Florida, when crafting your divorce settlement agreement. One such monumental, and very individual case is the case of Bair v, Bair. This case reached the Second District Court of Appeals, and the rulings can now be utilized by spouses in similar situations. Don’t waste time! Our writers will create an original "Whats Marital Labor and as a Business Owner: How Will it Affect my Divorce" essay for you Create order This specific case involved a business owner who had partial ownership of the business and also worked there full time. His employment ran the length of his marriage. Because he also held nonmarital assets in the company, this became somewhat complicated. Discussions on this case give Orlando Divorce Attorneys more information on business valuation and asset division. Understanding the Dispute The Background Before fully understanding the ruling, you need to be familiar with the background of the case. In 2012, Mr. Bair filed for divorce. Through their attorneys, the partners agreed on child custody and the division of the majority of the marital assets, including the home. The disagreement came when discussion moved to Mr. Bair’s business. Mr. Bair is part owner of a Florida Boat Dealership which was started by his father during his youth. It is still a family owned business, with all of the siblings owning shares. At the passing of his father, Mr. Bair inherited a larger portion of the company. He also worked at the business full time. The court was quick to agree that the business itself was ‘non-marital property’ and both parties agreed. The argument arose that because he had labored at the business throughout the marriage, any subsequent increase in business value resulting from his labor should be considered a marital asset. In 2017, the appeals court agreed- ruling that labor does qualify as a marital asset. The case was then returned to court to determine the correct amounts of the equitable distribution based on the new ruling. Equitable Distribution Florida Law mandates division of assets in an ‘equitable’ but not always equal manner. When calculating this division, the court looks at the following three areas: What assets qualify for equitable distribution due to being marital assets? What is the value of each asset to be distributed? What will be a fair distribution of the assets held within your marriage? There are some cases where this distribution is simple. If a couple has a joint bank account that contains $20,000 and the court has recommended equal distribution- then each spouse will receive $10,000 from that asset. Other assets, such as a family home, may not be as easy to distribute. Contact an Orlando DIvorce Attorney Financial considerations are some of the hardest parts of a divorce to understand. Using prior case law can help to guide you to an appropriate ruling that could be beneficial to your case. Discuss your case with your Orlando Divorce Attorney so they can help you to understand how these prior cases can benefit you.