Isolation of Recombinant Escherichia Essay Example for Free

Isolation of Recombinant Escherichia Essay One technique important in both genetics and biochemistry is the Polymerase Chain Reaction (PCR), first developed in the 1960s, and then automated in 1983. Current PCR technology was not developed until the discovery of thermostable polymerases, specifically Thermus aquaticus (Taq) polymerase (1). The protein Taq polymerase was first isolated from the extreme thermophile T. aqauticus, where extreme thermopiles are bacteria that live in temperatures at or above 45 °C. The Taq enzyme is a member of the DNA polymerase I family (2, 3). The interesting property of Taq polymerase is that it has a temperature optimum at 74-75 °C, allowing it the remain active in temperatures required for PCR double stranded DNA denaturation (3, 1) . The protein has an approximate molecular weight of 6263 kDa when isolated from T. aquaticus, and 94 kDa when isolated from recombinant Escherichia coli, and is still stable at temperatures of 93-95 °C, hence the thermostability of the enzyme ). Taq specifically lacks any proofreading activity in the 3’ to 5’ direction, and therefore has a relatively high error rate of single base mispairings of 1 error per Isolation of Recombinant Taq Polymerase for PCR 9000 nucleotides, as well as a frame shift error rate of 1 per 41,000 basepairs (5, 6). Taq polymerase has an activity that is highly dependent on the environment of which it is in as it is thermostable, and has differing activities at nearly all temperatures up to the point of denaturation. Taq specifically can add up to 1000 base pairs in length on a template in under one minute under typical PCR conditions. The enzyme has a specific activity of 200,000 units mg-1, and can add approximately form 60 nucleotides per second at 70 °C (7). The isolation of Taq is essential for the PCR reaction. The most important reason for Taq being used in PCR is the thermostability at high temperatures (95 °C). This allowed for the process of elongation, annealing, and denaturation to occur without the replacement of new enzyme, and thereby, was more efficient, faster, and cheaper because the reaction could be automated through the use of a machine known as a thermocycler which basically is just a machine able to change temperatures of an isolated environment rapidly (7). Prior to the discovery of Taq, PCR was done using Klenow fragments of E. coli DNA polymerase I at 37 °C. The lack of thermostability required replenishment of enzyme after each PCR cycle (8). One of the initial difficulties of Taq polymerase was the organism in which it was expressed in, T. aquaticus, as it was difficult to culture and produce large quantities of enzyme. E. coli bacteria were engineered to expressed the Taq polymerase gene to allow for retrieval of large quantities of enzyme ). The isolation of the Taq gene involved culturing T. aquaticus and then isolated the DNA of the cells through lysing, proteinase K addition, extracting of aqueous and phenolic phases, dialyzing of extractions, addition of SDS, and then centrifugation of solution to eventually retreieve the DNA of the organism as outlined in Lawyer et al., 1989. With the isolation of the 2401+ BP gene of Taq, the gene was incorporated into a 6.58 kbp plasmid (pLSG1). The gene was inserted 171 bp distal to the lacZÃŽ ± promoter/operator, and 109 bp distal to the BgII site, so the gene expression could be controlled through an inducible promoter. With the pLSG1 plasmid, the vector was introduced to E. coli bacteria to allow for plasmid uptake (4). Other experiments have been conducted towards the purification of Taq from recombinant E. coli. Specifically Engelke et al., 1990 developed a method for purfication of Taq. The E.coli strain 2 DH1 was used for the expression of the recombinant plasmid containing Taq polymerase. The bacteria were grown in 12 Litre batches of Luria Broth; using 1 mL of saturated DH1 culture and 80ÃŽ ¼g/mL of ampicillin. Isopropyl-1-thio-ÃŽ ²-Dgalactopyranoside (IPTG) was added to 0.5mM and the cultures were grown for 16-20 hours. The cells were harvested in 2.4 L of buffer A (50 mM TrisHCL, pH 7.9, 50 mM dextrose, 1mM EDTA) and collected via centrifugation, resuspended in Buffer A with 4mg/mL lysozyme and incubated at room temperature for 15 minutes. Buffer B (10 mM TrisHCl, pH 7.9, 50mM KCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Tween 20, 0.5% NP-40) was added and incubated in 180 mL fractions, for 60 minutes at 75 °C in a water bath. The mixtures were centrifuged at 8000 rpm for 15 minutes at 4 °C. Taq then precipitated with polyethyleneimine (PEI) at room temperature, then isolated through centrifugation and suspended in buffer C (20mM HEPES, pH 7.9, 1 mM EDTA, 0.5mM PMSF, 0.5% Tween 20, 0.5% NP-40) containing 0.25 M KCL. PEI eluatents were diluted in 50mM KCL and buffer C and applied to a 150mL BioRex 70 ion exchanger column, and then eluated using 200mM KCL. The protein was dialyzed for 12 hours against two changes of 1 L storage buffer (20mM HEPES, pH 7.9, 100 mM KCL, 0.1 mM EDTA, 0.5 mM PMSF, 1mM dithiothreitol, 50% glycerol. The experiment resulted in 40-50 mg of protein per litre of cell culture (9). The methods used in this experiment differed in certain key aspects. First, Engelke’s experiment made use of a higher concentration of ampicillin. The IPTG was added to the same concentration, but was added after cell growth up to an optical density of 0.700. Instead of a water bath at 75 °C, this experiment made use of an air incubator for the temperature requirements. Engelke’s experiment made use of PEI to precipitate Taq, while this experiment made use of 30g of (NH4)2SO4 per 100mL of supernatant. Buffer C was not used throughout this experiment, and no ion exchange columns were used. The dialysis procedure was done for twice as long with twice as many changes of solution per 6 hours. The changes made from Engelke’s experiment offers a different method for protein precipitation. The method used by Engelke made use of PEI which is an affinity precipitation method versus a salt prec ipitation method. The PEI Isolation of Recombinant Taq Polymerase for PCR method has the major drawback through the lack of selectivity, and can often precipitate nucleic acids as well (10). This is why the BioRex column needed to be used. Ammonium sulfate has the advantage that the precipitation can be controlled based on ionic strength of species involved, as well as has no negative effects on the activity of the target enzyme. Salting out also has the advantage that only native state proteins are precipitated due to the hydrophobicity involved with native state proteins (10). Buffer C was not required for this experiment as no BioRex column was required. This experiment made use of various techniques and methods including: SDS-PAGE, differential centrifugation, Western Blotting, real time-PCR (rtPCR), PCR, agarose gel electrophoresis, and dialysis. Two important techniques were PCR and rt-PCR. PCR does not allow for the quantification of DNA amplicons as it is an end-point PCR, but it does allow for confi rmation of template duplication along with measurement of base pair length. Amplification of primer would confirm the presence of a thermostable DNA polymerase. The following agarose electrophoresis helps to find amplicon size which can tell us the activity of Taq, as well as the specificity, as one template should only return one band in PCR (7). rt-PCR allows for a quantitative assessment of PCR, and therefore the kinetics of the reaction, as it detects the amount of amplicons produced in the reaction. The point at which the standard curve reaches threshold in cycle number gives information on the activity of Taq, as a more active sample of Taq reaches threshold earlier. Melt curve analysis also provides information regards DNA amplicons in solution (11). The purpose of this experiment was the test the methods for the isolation of PCR grade Taq polymerase from recombinant E. coli using differential centrifugation, salting out, and heat denaturation following lysation of cells to potentially improve isolation of Taq from past methods. The presence of Taq will be confirmed through Western blotting, and rt-PCR and PCR reactions along with purity will be assessed through SDS-PAGE. The activity of Taq will be found through rt-PCR and PCR. Finding the most efficient method for the isolation of Taq offers a valuable reagent source for any PCR reactions required. The isolation technique would also be applicable to any thermostable proteins. 3 EXPERIMENTAL PROECDURES Isolation of Taq Polymerase Luria broth (500 mL + 100ÃŽ ¼g/mL ampicillin) was inoculated with 50 ÃŽ ¼L of frozen Taq polymerase expressing E. coli cell stock. Incubation was commenced for 12 hours at 37 °C until the Optical Density had reached 0.700. IPTG (0.5 mM or 0.112g/L culture) was added and the culture was incubated for 12 to 14 hours at 37 °C. The 50mL of cells were then centrifuged (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810, and 5 mL of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1mM EDTA) was used to suspend the separated pellet. The solution was then centrifuged again (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810 and the pellet was once again suspended in Buffer A, with an additional 20 mg of lysozyme added. The reaction was incubated for 15 minutes at room temperature. Following incubation, 5mL of buffer B (10 mM Tris HCl, pH 7.9, 50mM KCl, 0.5% Tween 20, 0.5% NP-40, 1mM PMSF, 1mM EDTA) was added and incubated at 75 °C for 1 hour in a New Brunswick Scientific-Innova 40 incubator shaker series, and shaken by hand approximately every 5 minutes. The solution was then centrifuged (15000 RPM x 10 minutes at 4 °C) in a Thermoscientific Sorvall RC 6+ centrifuge and using a 603s Delta Range 30g of (NH4)2SO4 per 100mL of supernatant (8 mL of supernatant equivalent to 2.4g (NH4)2SO4 ) was added and incubated for 10 minutes at room temperature and shaken on the Innova 40 incubator. The lysate was then centrifuged again (15000 RPM x 10 minutes at 4 °C) in Thermoscientific Sorvall RC 6+ centrifuge and the resultant pellet was suspended in 2mL of buffer A. The solution was then dialyzed in a Spectra/Por membrane tubing set at 6000-8000 Da molecular weight selection in 1 L of storage Buffer (50 mM Tris HCl, pH 7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5 mM PMSF, 50% glycerol) for 24 hours at 4 °C changing the buffer every three hours. The dialysis solution was then diluted in a 1:1 ratio of storage buffer and stored at -70 °C until needed. Protein Concentration Determination A Bovine Serum Albumin Bio-Rad assay standard curve was prepared (0 –0.3 mg/mL) using Isolation of Recombinant Taq Polymerase for PCR a 1mg/mL stock solution and an Asys Expert Plus spectrophotometer set at 620 nm. Bio-Rad assay was run in triplicate using 20ÃŽ ¼L of protein dilution and 150 ÃŽ ¼L of diluted Bio-Rad Dye Concentrate. 10x and 100x dilutions of the sample prepared previously were made and 20ÃŽ ¼L were used with 150ÃŽ ¼L of diluted Bio-Rad Dye concentrate. The solutions were incubated for 10 minutes and absorbances were tabulated. sandwich was then assembled with an additional ice block in the transfer apparatus. The apparatus was run at 180mA overnight in a refrigerator and the membrane was then stored in TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and refrigerated. 24 hours prior to the primary antibody (Anti-Taq monoclonal Antibody (8C1)) addition, the membrane was blocked in 1 gram of Carnation nonfat dry milk (5% w/v) and 20 mL of TBST Buffer. The primary antibody in TBST with SDS-PAGE 5% w/v nonfat dry milk at a 1:800 dilution of A discontinuous polyacrylamide gel was antibody was added to the membrane and shaken prepared using a Mini-PROTEAN Tetra Cell for 1 hour at room temperature. The membrane was module. The casting stand was assembled for 1mm then washed three successive times for 15 minutes gel and filled with National Diagnostics 12% with TBST buffer at room temperature. The Resolving Gel (Protogel 2400 ÃŽ ¼L, Resolving Gel secondary antibody (Peroxidase-conjugated Buffer pH 8.8 1560 ÃŽ ¼L, dH2O 1974 ÃŽ ¼L, 30% w/v AffiniPure Goat Anti-Mouse IgG (H+L)) was then APS 21ÃŽ ¼L, TEMED 6ÃŽ ¼L), casted to 1 cm below top applied in TBST with 5% w/v nonfat dry milk at a of glass plate, and then 4% Stacking Gel (Protogel 1:2000 dilution of antibody and shaken for one hour 390 ÃŽ ¼L, Stacking Gel Buffer pH 6.8 720 ÃŽ ¼L, dH2O at room temperature. The membrane was then 1830 ÃŽ ¼L, 30% w/v APS 6ÃŽ ¼L, TEMED 3ÃŽ ¼L) was washed three successive times for 15 minutes with casted on top with a ten well gel comb. The TBST buffer at room temperature. The edges of the electrode set up was then submersed in 1 x Trismembrane were dried with a Kim and next 1mL of Glycine SDS Page Running Buffer. To each 20 ÃŽ ¼L Renaissance Western Blot Kit NEN Life Sciences of sample, 20 ÃŽ ¼L of Laemmli buffer (0.5 M TrisProducts, Cat. No. NEL 101 luminol reagent with HCl, pH 6.8, 4.4% w/v SDS, 20% v/v glycerol, 2% 1mL of oxidizing reagent were mixed together and v/v 2-mercaptoethanol, 10 mg/mL bromophneol then applied to the membrane. The membrane was blue) was added and boiled for 3 minutes and then then imaged with an exposure time of 40 seconds cooled for 5 minutes on ice. To one well 7ÃŽ ¼L of using AlphaEaseFC software. New England BioLabs Inc. Prestained Protein Marker (7-175 kDa) was added. To the following PCR/agarose gel electrophoresis wells 15 ÃŽ ¼L of standard Taq polymerase was added, A master mix for PCR was prepared (1x PCR along with 20ÃŽ ¼L of six different samples, with the buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, fifth being prepared the previous year with the same 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 method of isolation as outlined previously. The gel ng Template DNA and Nuclease-free PCR water) was run at 200 Volts for 40 minutes, incubated in and 22.5 ÃŽ ¼L of master mix and 2.5ÃŽ ¼L of Taq fixing solution overnight and then stained with Bio- sample, the standard, or the Taq prepared a previous Safe Coomassie Blue for one hour at room year were added to PCR tubes and centrifuged temperature under agitation. The gel was then briefly on a Fisher Scientific Accuspin micro 17 analyzed used AlphaEaseFC software. just briefly using 1.5mL eppendorf tubes with no caps to contain the PCR tube. The PCR tube was Western Blotting then added to T3 Biometra Thermocycler and Using the method described above for SDSdenatured at 94 °C for 3 minutes and then 35 cycles PAGE, a SDS-PAGE gel was taken prior to fixing. of PCR with the denature 94 °C for 45 seconds, The gel was then transferred to transfer buffer anneal 55 °C for 30 seconds, and extension at 72 °C (20mM Tris-HCL, pH 8.0, 150 mM Glycine, 20% for 1.5 minutes. The sample was then incubated at Methanol). Immobilon-P transfer membrane with 72 °C for 10 minutes and then temperature was 0.45 ÃŽ ¼m pore size and Whatman paper were cut to maintained at 4 °C. The samples were then stored at the size of the gel. The membrane was wet with -20 °C until agarose gel preparation. A 1% agarose 100% methanol, then transferred to MilliQwater gel w as prepared through 1.5g of agarose (Sigma and soaked for several minutes. A standard blotting No. A-6877 Type II) to 150mL of Tris-Acetate4 Isolation of Recombinant Taq Polymerase for PCR EDTA (TAE) buffer. The solution was microwave for 1 minute and mixed until in solution. Once cooled to 60 °C, 7.5 ÃŽ ¼L of Biotium Gel Red Nucleic acid stain was added and mixed. The solution was then poured into the electrophoresis tray; a comb was installed, and set at room temperature. One Litre of 1x Tae buffer was prepared through dilution of 50x TAE buffer and then the solution was poured onto the electrophoresis tray to cover the gel in 1mm of buffer. 20 ÃŽ ¼L of PCR product prepared previously and 4ÃŽ ¼L of Gel Red dye were mixed and 20ÃŽ ¼L of each sample, the standard, and Taq prepared the previous year and Invitrogen life Technologies 1 Kb DNA ladder Cat. No. 15615016 was run at 150 Volts, 100 mA for one hour (or until dye reached the bottom of the gel). The bands were then visualized under 300 nm light and fluorescence was measured at 590 nm. The gel was analyzed using AlphaEaseFC software. concentration of the sample Taq was 1.88 + 0.11 mg/mL. The solution of proteins was not pure Taq as confirmed by the SDS-PAGE (Fig. 2) as various proteins created distinct bands (B to K excluding E). The standard Taq revealed only one band (A), indicating band E was most likely belong to Taq, as it was the darkest band in the gel. An analysis of the molecular weights of the bands through electrophoretic mobility (Tab. 3) showed the standard Taq having a molecular weight of 115.2 + 14.6 kDa, and the likely band (E) had a molecular weight of 113.4 + 14.3 kDa. There was a distinct distortion in the bands of the SDS page in all lanes with the exception of the standard Taq and the 2011 Taq (Fig. 3). The distortion is of a smile. The overall gel also has a large distortion, but of a frown. It would appear there was a similar protein to D E and F present in all samples, including the 2011 sample. The standard Taq did not contain the bands. Re al Time PCR The Western Blot (Fig. 4) revealed distinct A master mix for PCR was prepared (1x PCR bands; however, there were more than one band in buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, each lane with the exception of the standard Taq. 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 Two distinct bands were present in 5, Taq, and 2 (b, ng Template DNA and Nuclease-free PCR water). c). The lanes of * and ? contained several bands To PCR tubes, 22.5 ÃŽ ¼L of Master Mix and 2.5 ÃŽ ¼L also. The overall gel also expressed a slight color of Taq sample or the standard Taq were combined, banding along the solvent front edge which is mixed through vortexing and then centrifuged with shown in both Fig. 3 and 4. The 2011 lane did not a Fisher Scientific Accuspin micro 17 just briefly appear to have any Taq present, as no band was using 1.5mL eppendorf tubes with no caps to distinguished. The entire ladder expressed some contain the PCR tube. The Taq samples were antibody activity. prepared in triplicates. 20ÃŽ ¼L of each sampled were The real time-PCR revealed a threshold reached then transferred to a 96-well PCR plate and then at 20 cycles, with the vast majority occurring at 24 sealed. The well was then placed in a BioRad CFX cycles. The melt curve showed an approximate connect Real Time System using the programing of melting temperature of 81 °C (Fig. 7). enzyme activat ion (95 °C, 30 seconds, 1 cycle), 40 The agarose gel electrophoresis revealed one cycles of Denaturation (95 °C, 1 second) and distinct band at approximately 5883.5 base pairs in annealing/extension (60 °C, 5 seconds), with a melt length. The brightest bands, and therefore the curve of (60-95 °C in 0.5 °C intervals, 3 seconds per highest quantities of Taq enzyme were found in the step, 1 cycle). The samples were then analyzed std., 2 and 4. When the base pairs specific activity using AlphaEase FC software. of the enzyme was calculated it was found to be 834.5 + 63.9 bp/min/ÃŽ ¼g of sample, or 3922.3 + 192.9 bp per minute. RESULTS The results of the Bio-Rad assay on the sample of Taq polymerases diluted to 10x and 100x revealed that the 10x dilution was far to concentrated and fell outside the linear curve of the Bio-Rad assay. The retrieval of protein from the Luria broth was found to be 300.8 + 17.7 mg protein per L of Luria broth. These results (Tab. 1) suggest the protein 5 DISCUSSION Through the analysis made through SDS-PAGE, the MW of the standard Taq was found to be 115.2 + 14.6 kDa and 113.4 + 14.3 kDa. This is different from the accepted literature value of 94 kDa (9). Even with error correction, the prot ein did not fall Isolation of Recombinant Taq Polymerase for PCR within the range of the accepted literature value. In total, the two proteins differ by 23% and 21% without error correction, or 21.2 kDa and 19.4 kDa respectively. In comparison to one another, the two bands have essentially the same molecular weight, indicating whatever error occured in the gel was equivalent on both the standard and the isolated Taq. One explanation for the difference in the molecular weights may be explained through the quantitiy of protein used. The darkest and thickest band ( E, fig. 4) likely belongs to the Taq protein. To get a more defined band, a dilution would be effective in making a higher resolution band (12). The amount of protein isolated per volume of Luria broth was determined to be 300.8 + 17.7 mg per L of Luria Broth. Quite obviosuly, there are issues both with the heating of the gel, and distortion of the bands into â€Å"smiles†. The distoration of the gel likely was caused by unequal heati ng of the gel causing the center of the gel to be hotter than the peripheries, as the walls of the apparatus act as heat sinks (13). The uneven heating can be removed by switching to a lower voltage for a longer period of time (12). The distortion of the protein bands within the individual lanes produced a smile structure. The distortion was likely caused by either an overloading of proteins, which can be solved by dilution of the protein sample, or was due to salt conditions of the loading sample. This step could be fixed through extra steps of dialysis to decrease salt content of the loading sample. (14). One final issue with the SDS-PAGE gel was the distance between bands. The target molecular weight was near 100 kDa, so the concentration of the gel could be decreased to allow for a higher resolution of the higher molecular weight proteins, or allowed to run for a longer period of time (14). A purity assessment of the isolated Taq enzyme can be made through the SDS-PAGE gel (fig. 2). Distinct banding occurs in ten different bands on the Taq lane, with 9 being distinct from Taq protein (E). This highlights that there were infact multiple proteins still present in the Taq solution. This would indicate that the heat shock portion of the methods was insufficient in denaturing all of the proteins in the E. coli, allowing for precipitation upon salting out. This is based on the extra protein banding only occuring for the Taq polymerases prepared for this experiment. A factor that could have also played a role was the incubation at 75 °C was continually 6 interrupted through the need to shake the reaction vessel thereby lowering the temperature of the solution. This was due to mechanical difficulties of the equipment. It would be best to find a working New Brunswick Scientific-Innova 40 incubator shaker series to improve the protein isolation. To decrease the protein impurities, an increased heat cycle could be implemented, as Taq is thermostable at 75 °C, and could sustain structure at that temperature for long durations (7). The ammonium sulfate salting out would be mor e efficent after an increased heat cycle as even fewer native state proteins would remain (10). Another method to decrease impurities would be to add a purification step using another specific property of Taq polymerase. This could be the isoelectric point. This could be done through ion exchange columnsor isoelectric focusing (12). The extra isolation step would significantly decrease the impurities, and increase the specific activity per mg of protein of sample.The impurities were likely a result of other proteins present in E. coli bacteria lysate that were relatively thermostable, as those proteins would be most probable (9). The isolation of Taq can be confirmed through the Western Blotting and PCR reactions (Fig. 4-7), as a distinct band in the Western Blot, and measureable amplicon replication in the PCR and rt-PCR. In the standard of Taq of the Western blot (Fig. 4) there is a distinct band. The same band in the channel containing the isolated Taq can be seen. The band occurs in the same relative vicinity as the Taq molecular weight band in the SDS-PAGE (Fig. 2) so would fit best fit the Taq enzyme. The banding of the blot shows a common band across all lanes that line up with the standard Taq, emphasizing the isolation of Taq. There is a hesitation in confirmation of Taq due to the extra protein banding in the prepared fractions, as these bands were not seen in the standard Taq. The banding would suggest proteins transferred from the gel to the membrane and was still able to bind to the primary antibody or secondary antibody. There are various possible explanations for this. First and foremost, the banding occurred in areas wherever protein was present (ladder and lanes). This would indicate lack of specificity in the primary antibody which is intended to only find full sequence Taq and bind to it (15, 16). Another problem may be due to lack of blocking solution binding to the membrane, or Isolation of Recombinant Taq Polymerase for PCR excessive washing removing blocking solution from the membrane. A final possible explanation may be binding of the secondary antibody to membr ane bound proteins with the exception of casein (the blocking protein used) (15, 17). Antibody specificity can be corrected by finding a new antibody, lack of blocking simply requires longer blocking periods or increased blocking solution concentration, and washing can be minimized to see resultant effect on the membrane. Each of the possible problems with the Western Blot would have to be tested by altering the procedure used above by one method (washing, antibody, blocking solution). The PCR results show template replication through thermocycling, which indicates the presence of thermostable DNA polymerases in the PCR tube. From this, it can be conferred that Taq polymerase was indeed isolated. Further confirmation could be made through further purification of Taq. This could be done through 2-D SDS-PAGE vs Isoelectric point electrophoresis using the isoelectric point of Taq and using the bands emphasized as Taq, and a lower concentration gel (12). Another method would be to analyze the gel bands through other methods such as mass spectropscopy or NMR (18). There wa s distinct differences between three sets of Taq polymerases: the standard, the sample prepared in the previous year, and the sample produced in this experiment. Most distinctly the proteins differ with respect to SDS-PAGE gels. Quite obviously, the purest of the enzymes was the standard Taq, followed by the 2011 sample, and the sample prepared in this experiment. The sample prepared through this experiment had a high amount of a salt concentration and resulted in distorted bands, along with numerous other proteins present in the sample. The enzymes also differed with respect to the Western Blot (Fig. 4). The 2011 sample failed to return 2 ° antibody response, indicating lack of Taq polymerase, or lack of primary antibody binding, while the standard and experimental sample both had representive banding. There may have been excessive blocking or drying of the lane containing the 2011 Taq, as the SDS-PAGE shows a representive band in the region of Taq, that is the darkest band in the lane (15). The protein concentrations as determined through the Bio-Rad assay (Tab. 1, Fig. 1) returned 7 drastically different results. The two protein concentrations differed by 2x concentration. The easiest explanation of thi s result is the 10x dilution was insufficient in reducing the absorbance to within the standard curve. Due to the absorbances being above the standard curve, the results are invalid, as the region in which the curve is linear is up to 0.5mg/mL (19). The 100 x dilution returned a result of 1.88 + 0.11 mg/mL. This coroborates the SDS-PAGE findings as the protein was not excessively overloading the lane. The SDS-PAGE could have been further diluted, but the concentration used was sufficient for the purposes of the experiment. In an analysis of the PCR results (fig. 7), the brightest fluorescence bands occurred in the std., 2 and (4/Taq) lanes. This would indicate the highest activities occuring in these lanes. When compared to the western blot, the darkest banding of regions of Taq (5,?,*) returned the bands with less fluorescence. This result shows that the amount of enzyme may inhibit the PCR reaction as the the bands with the highest recoveries returned the lowest fluorescence. With an assessment of the basepair length, reaction time, and amount of enzyme used, an approximately activi ty of 834.5 + 63.9 bp/min/ÃŽ ¼g of protein, or 3922.3 + 192.9 bp per minute. In comparison to the literature values of the protein, this is slightly above the 60 base pairs per second value, however, that was at 70  °C (7). The rt-PCR returned a consistent melting temperature of 81 °C (Fig. 6)for all amplicon samples indicating the lack of a primer-dimer formation. Threshold was initially reached at 20 cycles (Fig. 5), which an RFU value of approximately 9000. This indicated a high activity of the taq polymerase used, at least above 1.25 Units (20). Both PCR assays agree with one another. There was no primer dimer formation noted on the agarose gel, or the melt curve analysis. There was a high activity of the enzyme sample isolated as found through the bpmin-1 and cycle # of reaching threshold, however, between the two assays, the rt-PCR has the significant advantage of time, and no electrophoresis required. Currently, Taq is widely available and would likely be cheaper to simply purchase commercially. This experiment does however outline a method for thermostable protein isolation which could be used for the more recent and more valuable thermostable enzymes (Pyrococcus furiosus Polymerase) which Is olation of Recombinant Taq Polymerase for PCR are superior to Taq in both thermostability, and error rate due to proofreading ability (21). Overall, the purpose of the experiment was met. Taq was indeed isolated from a culture of recombinant E. coli. This was confirmed through the Western Blotting, and thermostable DNA activity in the PCR and rt-PCR. The purity was assessed and found to be below that of the methods used by Engelke et al., 1990. The purity could be increased through use of a cation exchange column (9). The length of heat denaturation and an automatic heat controlled shaker would help to remove excess proteins and improve purity. The length of dialysis time would need to be increased for less band distortion in SDS-PAGE, and either more selective primary antibody, increased blocking or decreased washing would be required for improved Western Blotting. For further experiments, it is suggested testing the new method modifications, and or implementing recombinant Pyrococcus furiosus Polymerase.

The Successes And Failures Of The Obama Presidency Politics Essay

The Successes And Failures Of The Obama Presidency Politics Essay President Obama is seen as a successful President according to the website Politifact.com, out of the 502 promises he made during the 2008 election campaign he has so far kept 134, compromised on 41, broken 40, stalled on 69 and still has 220 in the works. He may, therefore, be seen to be a successful President. However, this was not reflected in the mid-term elections where the Democrats lost a considerable number of seats within the Senate and lost their majority within the House. In this essay I will assess the successes and failures of the Obama presidency against what he promised during his election campaign of 2008 and during his subsequent State of the Union address. I will also judge some of his successes and failures by reference to opinion polls from various news outlets, particularly whether the public approved of his handling of certain situations such as unemployment and the BP oil disaster. I will first assess his domestic successes and failures then his foreign policy successes and failures, while assessing what it means to his presidency specifically and the American political system. President Obama knew when he came into office that his first priority was to fix the economy after the recession of 2008-2009. He introduced the American Recovery and Reinvestment Act of 2009 which provided a stimulus to the country; it was split into three parts at a cost of $787 billion. One-third in tax cuts and tax credits to help overcome the hardships of the recession, such as an increase in food stamp benefits and unemployment benefits; one-third on the infrastructure of the country, which included projects on transport and highway and bridge construction and repair. Finally, one-third on state stabilization to stop the states cutting essential services that the public need, such as teachers.  [2]  President Obama also bailed out the car industry, specifically Chrysler and GM, at a cost of $17.4 billion.  [3]  The necessity to save the car industry was evident as it could have led to a double dip recession. GM alone employed 300,000 workers within the United States, an d if it was to fall into insolvent administration it would have had a large impact on the economy. President Obama also initiated Cash for Clunkers scheme in which owners of cars could trade them in for more fuel efficient cars. This was very popular as the initial $1 billion in subsidies for this program was supposed to last for 90 days but actually only lasted for 9.  [4]  He also brought in measures to help nine million homeowners facing foreclosure on their homes and who could not afford to repay their mortgages. The purpose was to offer mortgage companies subsidies and incentives to help troubled home owners.  [5]   Obama also brought in regulation to control the banks after they started making profits again. He stated: We are not going to let Wall Street take the money and run  [6]  and imposed conditions before the banks could escape George W Bushs Troubled Asset Relief Programme (TARP) in which money was given to the banks in total costing $700 billion.  [7]  Even though the stimulus was intended to create jobs, they were not planned to be created until 2010-2011. In 2009 unemployment was 10% of the workforce and as of March 2011 the unemployment rate still hovers around the 10% mark.  [8]   Overall Obama has kept most of his promises regarding the economy, with very few promises broken.  [9]  However, in a poll of how he handled the economy his approval rating is only at 45% (January 2011) with 54% disapproving.  [10]  This shows that the public thinks he is handling the economy quite badly. Critics have pointed out that he has done well on the economy with Nouriel Roubini in an article in the Financial Times pointing out that he has inherited the worst economic crisis since the Great Depression and that the stimulus did the right thing early and avoided another depression  [11]  Without the stimulus Jonathan Alter makes the point that 8.5 million workers would have lost their jobs. He has done well in regulating the banks and TARP with the loans with added interest that were made having now been paid back. The figures are seen as impressive as instead of $700 billion being owed to the Government, the Government is owed just under $100 billion. The Clash for Clunkers programme is estimated to have saved 120,000 jobs.  [12]  The auto industry has also been greatly improved creating 55,000 new jobs and GM paying back its bailout money five years ahead of schedule.  [13]  Obama recalls that It wasnt an easy callà ¢Ã¢â€š ¬Ã‚ ¦But we decided that while providing additional assistance was a risk, the far greater risk to families and communities across our country was to do nothing.    I knew this wasnt a popular decision. But it was the right one.  [14]   The way he dealt with the housing crisis was seen to go not quite so smoothly as of the 9 million homeowners the plan hoped to assist, only 3 million were actually helped. This was due to the fact that the legislation was directed at people who the Obama administration saw as having a good reason to stay in their homes; these people often worked full time and had received sub prime mortgages. The legislation helped stop house prices falling by the predicted 20% and instead increased them by 5% although house prices are still of considerable concern to the US economy.  [15]   Where Obama has failed is in job creation. It is at its worst since 1982. Newt Gingrich of the Republican Party commented If were still at 9 or 10 percent unemployment in 2012, this is a one term president no matter how articulate he is. The unemployment rate is furthermore one of the factors why the Democrats failed in the mid-term elections losing control of the House.  [16]  As Alter states, for Obama even to be considered a success he must bring down unemployment.  [17]  Obama has been able to pass all of his economic legislation quite easily due to his previous super majorities within the House and the Senate after the election of 2008. However, as he has now reached the mid-terms and the Republican party have a majority hold within the House, he will have a much difficult time as the Republican party are likely to resist his legislative proposals. This is becoming apparent in the differences in Congress over this years budget proposals and points to the difficulty when any president is faced with his party being in a minority within Congress. One of Obamas major pledges was to reform the ailing healthcare system, as it would greatly increase the countrys debt. Healthcare spending has grown to such an extent that it is predicted to rise from one-sixth of GDP in 2009 to one-third in 2035, and by 2080 nearly half of U.S. GDP would be spent on healthcare according to the Congressional Budget Office.  [18]  Healthcare premiums had also doubled and the numbers of uninsured Americans would surpass 50 million. Families have told of being bankrupted by paying for the medical care, and of the bad treatment from insurance companies.  [19]  Obama and a Democrat Congress implemented a healthcare plan which contained new federal regulation of insurance, help for states on Medicaid, an increase in the affordability of healthcare and higher fees on employers who do not provide insurance.  [20]  This was met with fierce opposition from some on the right of the political spectrum such that no Republican congressmen voted for it , mainly encouraged by those from the extreme right of the political spectrum, such as the Tea Party movement. The healthcare plan was seen as a success as it was one of Obamas key campaign promises to ensure that some sort of healthcare plan became law, and he again mentioned in his 2010 State of the Union Address that he wanted to implement some healthcare reform.  [21]  He got his wish with the Health Care and Education Reconciliation Act of 2010. Although seen as a success in that he got the Democratic Congress to pass the necessary legislation, it proved unpopular with the public with one poll giving 38% approving of the healthcare plan and 53% opposing it.  [22]  However, different news organisations reported different numbers at separate times. President Obama had to rely on his presidential powers of persuasion and bargaining to get this legislation through Congress, as he influenced Senators, House members and even tried unsuccessfully to get Republicans on his side. At various times he tried to influence public opinion that such legislation was necessary. The legislation is also subject to numerous court challenges with Judge Roger Vinson in the Federal District of Pensalcola, Florida ruling that the whole legislation was void, not just the provisions for compulsory insurance.  [23]  It is likely that the Supreme Court will eventually be called upon to rule on the legislation. The importance of a president leading on controversial legislation and using all of his powers of persuasion to carry Congress and the public with him is demonstrated by the healthcare legislation. President Obamas other domestic policy initiatives include bringing in legislation on the repeal of dont ask dont tell, which stops the military from infringing on the rights of homosexual citizens if they choose to join the military; his education reforms which gave the power to close poor performing schools (which Bill Gates rated him a A for); and the equal pay legislation which promises equal pay for jobs that have equal standing. Obamas biggest domestic disaster came with the BP oil spill. Obama could be said to have handled the BP oil spill poorly as he was slow to respond and criticised for not being more emotive on the issue, saying in an interview that This is not a theatre.  [24]  Polls at the time showed a low approval rating of just 41%.  [25]  Nevertheless once the issue was resolved, it was quickly forgotten about by the media and the Government. President Obamas foreign policy is one of both successes and failures. One of Obamas main campaign promises was to withdraw from Iraq which he successfully completed on the 31 August 2010 with only a handful of troops staying in Iraq to advise Iraqi forces.  [26]  Afghanistan was the most controversial foreign policy decision during his presidency with Dick Cheney accusing him of dithering. Obama after much deliberation sent 30,000 more troops into Afghanistan with the decision to withdraw some troops in summer 2011. The President of Afghanistan accepted cash bribes from Iran which caused embarrassment to the Obama administration and General McChrystal and his staff criticised the Commander in Chief in a magazine article, calling him unimpressive and uncomfortable and intimidated. He was dismissed and replaced by General David Petraeus. Obama has made positive steps to improve U.S.-Russia relations which saw a dispute with the Bush administration over the missile defence system i n Eastern Europe which Russia strongly opposed. Obama scrapped this idea and began talks with the Russians and these resulted in good levels of cooperation with Russia over issues such as Iran and North Korea, and the Strategic Arms Reduction Treaty (START) which was approved by the Senate and became effective on the 5 February 2011.  [27]  28He made the aid funding to Pakistan conditional on its controlling terrorist activities. Obama has been successful in banning torture as an interrogation technique. He has been criticised from the right as being slow in supporting the Iranian uprisings in 2009. However, he was said to have exercised restraint on the issue as he did not want the Iranian government claiming that it was an American sponsored uprising.  [29]  Again he was criticised over his handling of the popular uprisings for democracy in 2011 in the Arab nations, such as Egypt and Tunisia, with Christopher Hitchens calling the slow response to these uprisings as a dithering responseà ¢Ã¢â€š ¬Ã‚ ¦[which has] been both cynical and naive.  [30]  The same too could be said about Obamas response to the Libyan rebellion. However, it is clear that Obama wanted a multilateral response, particularly from the Arab League, and UN approval before rushing into another war.  [31]   Obama has tried to enhance relations with other countries, unsuccessfully in the case of Iran and North Korea, but successfully with Europe, China and more particularly with Russia. His meeting with the Russian President in June, 2010 at the White House was said to mark the highest point of Russian-U.S. relations since Bill Clinton went to Moscow to mark Victory Day.  [32]   Obama is said to be badly at odds with Israel with the Financial Times stating: Mr. Obama initially played tough with Mr. Netanyahu but then backed downSettlement expansion puts out of reach an agreement on borders The US veto at the UN badly damaged Mr. Obamas credibility. The resolution, after all, reflected the White Houses own stated position..Mr. Obama must choose between prevarication and the chance to sustain US influence in the worlds most troubled and strategically vital region.  [33]   His biggest failures to date are Afghanistan and Guantanamo Bay. Obama acknowledges that progress in Afghanistan is limited and fragile although there has been success in resisting the Taliban and Al-Qaida.  [34]  It is still unpopular with the American public with just 31% of the American public thinking Afghanistan is worth fighting for.  [35]  Obamas most problematic issue comes with Guantanamo Bay with the website Politifact.com listing it as a broken promise. Congress and the President remaining at odds over where to put the prisoners due to the refusal of Congress to allow detainees to be imprisoned within the United States . On the 7 March 2011 Obama signed an executive order making a number of changes to his policies, with a number of news outlets noting that it is an acknowledgement that he could not keep his campaign promise of closing Guantanamo Bay.  [36]   President Obama has overall seen far more successes than failures during his Presidency although he has yet to convince the American people. Although this could all change with the losses during the mid-term elections as he will find it a lot harder to pass his legislation. His recent reforms are under threat from the Republicans, such as Healthcare and environmental legislation which they want to scrap.  [37]  The President has been nicknamed no drama Obama but as the Financial Times states this is often perceived as him having a sense of aloofness and on the national and international stage a sense of unwillingness to lead.  [38]  At the end of last week 64 senators, 32 Republican and 32 democrats, asked the President to lead on a comprehensive plan to deal with fiscal policy. As the Financial Times points out in a leader, Mr Obama should be embarrassed that the Senate finds it necessary to ask him to do his job.  [39]  The importance of a president providing leadership is again emphasised. His priorities should be to deal with the issues facing Afghanistan, the US debt burden and the unemployment crisis, because as Newt Gingrich stated he will have a hard time becoming re-elected in 2012 if he does not solve these issues. These issues will no doubt have to be grappled with by any president elected in 2012.

black people :: essays research papers

Some black people think everyone is messy! Others can't stand us. Yes I prefer to be called African American than black! Only once in a lifetime will a new invention come about to touch every aspect of our lives. Such devices changed the way we manage, work, and live. A machine that has done all this and more now exists in nearly every business in the United States. This incredible invention is the computer. Computers are one of the most important inventions ever. If computers had not been invented, technology would not be developed to its current state. Since the computer invention, society has changed severely. Computer technology is so helpful, that it is even used to create newer, better computer equipment. Almost everything today is linked in some way, to a computer. Until this decade, computer technology was non-existent in public school systems. Computers are valuable to schools for many reasons. They are good for studying and research, if the sites are indeed factual. Computers supply a way to type papers, they can be used for business classes, and it can provide children with something they may enjoy using. If students are interested in what they are doing, they will do it better. Computer programs are also very helpful to business classes. Accounting classes and computer related fields of study must have computers to be current with today's business. Accounting today is all computerized. It is necessary to familiarize students to the functions of various programs if they are going to go into one of those fields. Computers have also made communications easier than ever. Today, e-mail is beginning to replace the ordinary post office and telephone as a way to keep in touch. E-mail provides the best of both worlds; it is instant and free. Before e-mail, one would have to send a letter that would take days to arrive, or they would have to use the telephone, which would cost money if the calls were

Essay --

There have been many instances in history where people have overthrown their current government in order to create a new, better government in its place; we can see this especially in the American and French Revolutions and even in the Communist Revolution in Russia. Revolution is â€Å"a forcible overthrow of a government or social order in favor of a new system.† This type of behavior is intended by Thomas Paine and John Locke—who believed in recovering natural rights for all—but also by Karl Marx who strongly believed all institutions should be broken down completely; these influential leaders had opposite ideas for the future of not only their nations but the nations of the world, they both had supporters to spread and implement their ideas in society. Although both sides led significant revolutions, ultimately the revolution that recuperated self-evident truths prevailed over the revolution that’s intent was to destroy religion, family, and all other institutions. Karl Marx, the founder of communism, strongly believed that his revolution would be the last; he believed it would incorporate the whole world. According to Marx, four steps would take place for there to be total communism: feudalism, liberalism, socialist revolution, and then communism. Liberalism (and capitalism) would take the place of feudalism and would enrich the few at the expense of the working poor. After this, the socialist revolution would ensure that the proletariat (working poor) would seize all private businesses and redistribute the wealth equally among all. Only after this took place, could communist truly take reign at the global level. â€Å"The modern bourgeoisie society that has sprouted from the ruins of feudal society has not done away with class anta... ...pendence in the French revolution. The Declaration of Independence has served an important role in history as well as in modern society and uncovers the American values we still live by. Despite the ruthless attempt for complete communism, communism failed. It proved to be inefficient. Self-evident truths proved to be more successful in government and have lasted the test of time. Marx failed to understand the importance of institutions in society and the necessity of human values. By destroying family and religion, Marx is also destroying human desire to succeed. Society is always aiming to improve. If all were equal in the economic sense, society would be at a plateau because competition would seize. People want to work toward some goal or for a cause such as family or property. To recover self-evident truths is to ensure the growth and proficiency of society. Essay -- There have been many instances in history where people have overthrown their current government in order to create a new, better government in its place; we can see this especially in the American and French Revolutions and even in the Communist Revolution in Russia. Revolution is â€Å"a forcible overthrow of a government or social order in favor of a new system.† This type of behavior is intended by Thomas Paine and John Locke—who believed in recovering natural rights for all—but also by Karl Marx who strongly believed all institutions should be broken down completely; these influential leaders had opposite ideas for the future of not only their nations but the nations of the world, they both had supporters to spread and implement their ideas in society. Although both sides led significant revolutions, ultimately the revolution that recuperated self-evident truths prevailed over the revolution that’s intent was to destroy religion, family, and all other institutions. Karl Marx, the founder of communism, strongly believed that his revolution would be the last; he believed it would incorporate the whole world. According to Marx, four steps would take place for there to be total communism: feudalism, liberalism, socialist revolution, and then communism. Liberalism (and capitalism) would take the place of feudalism and would enrich the few at the expense of the working poor. After this, the socialist revolution would ensure that the proletariat (working poor) would seize all private businesses and redistribute the wealth equally among all. Only after this took place, could communist truly take reign at the global level. â€Å"The modern bourgeoisie society that has sprouted from the ruins of feudal society has not done away with class anta... ...pendence in the French revolution. The Declaration of Independence has served an important role in history as well as in modern society and uncovers the American values we still live by. Despite the ruthless attempt for complete communism, communism failed. It proved to be inefficient. Self-evident truths proved to be more successful in government and have lasted the test of time. Marx failed to understand the importance of institutions in society and the necessity of human values. By destroying family and religion, Marx is also destroying human desire to succeed. Society is always aiming to improve. If all were equal in the economic sense, society would be at a plateau because competition would seize. People want to work toward some goal or for a cause such as family or property. To recover self-evident truths is to ensure the growth and proficiency of society.

Essay --

Tricked by Inferno (A critique on how Dante’s Inferno can make you do the morally correct thing) As the great Francois de La Rochefoucauld, â€Å"The intellect is always fooled by the heart.† When it comes to doing the morally correct thing, Dante’s Inferno is the text that scares people to do what they are supposed to do. By saying what will happen if a person were to go to hell, this will scare people into doing the right thing. As Tim Keller said, â€Å"Sin removes us from that aspect of his power that sustains and supports us. It is to us as water is to a fish-away from it our life slowly ebbs away.† In Dante’s Inferno he uses many tools to scare people into acting as they morally should, three of these critiqued tools include: pain, suffering, and misery. To begin, pain is an ideal threat that Dante provides in the idea of hell. When the thought of hell arises, one of the very first things that comes to mind is pain. The pain of realization that you are going to spend the rest of eternity in purgatory is very excruciating. â€Å"May you weep and wail for all of eternity.† (pg.81) This quote from Dante’s Inferno states just what kind of pain there will be if a person went to hell. Weeping knowing that for the rest of eternity there will be only pain, and heartache. â€Å"Heartbreak is like one big emotional pain but it also seems to spark off hundreds of other emotions. We hate the feeling of heartbreak, and yet we find ourselves compelled to go over and over memories, ideas or fantasies which make the feeling worse. â€Å" As Dr. Edward E. Smith states, heartache is one of the worst feelings that a person can face. After realizing the kind of heartache and pain that going to hell can cause, people will be more tempted to act morally correct. Hell has... ...for no reason, but to bring themselves up they deserve to be punished. Hurting people is no way to make you feel better. Therefore they should get a taste of their own medicine. The people in this circle will be stomped on by a giant, over, and over, for all of eternity. They will be â€Å"be-littled† literally, after lifting the egos of all of the people that they were mean to during the time that they were alive. Circle 6 (Rapists and Murders) This is the worst crime that person could ever commit. These people will be shot over and over for the rest of eternity. They did the worst thing humanly possible. Raping someone and or murdering them is the worst thing a person can do. Taking everything that person has is the worst thing that someone could do. By being shot repeatedly they will be able to feel the pain that they have caused other people while they were alive.

Comparison Between E-Business and Traditional Business

INTRODUCTION Electronic Business (E-Business) is a perplexing practice due to the numerous aspects it involves. In today’s rapidly changing environment, organizations adopt E-Business to respond to several business drivers. The progressions of the macro-environments are creating innovative business environments, in which E-Business is considered a normal practice. This paper attempts to model the business environment and evaluate its competitive characteristics by comparing the traditional business with E-Business. Kreplin.K, et al (2000), identified â€Å"Reality† and â€Å"Virtuality† terms; these terms differentiates traditional business from E-Business. According to Kreplin. K, et al (2000), E-Business is based on a virtual (digital) business process with a virtual agent, and virtual product. Traditional Business is a physical business process with respect to the macro-environments. The macro-environment components can influence the way entrepreneurs use the internet to coordinate export businesses. Analysis of the macro-environment comprises of cultural, economic, competition, political and legal factors that affect the way business transactions are made today.CULTURAL CHALLENGES In the cultural dimension, traditional business entrepreneurs will face a major challenge. In this case, infrastructure cost is what worries traditional businesses of today. As opposed to E-businesses, online business transactions incur minimal cost (Robertson. B & Sribar. V, n. d. ). Whereas, traditional businesses will have to incur a significant amount of cost in order to remain competitive in the market. This is due to the fact that there is a paradigm shift towards a more innovative market alongside the consumers.Through the report it is assured that in the long term infrastructure cost will be the key obstacle for traditional entrepreneurs as newer technological innovations take place (Robertson. B & Sribar. V, n. d. ). Opposing to the traditional method of business, E-businesses also have their share of cultural challenges. Firstly, entrepreneurs will experience a change in the nature of workforce (Parreiras. F , n. d. ). According to Heerwagen. J, Kelly. K, Kampschroer. K (2010), the structure of work is now more cognitively complex, team-based, nd time pressured. This movement causes entrepreneurs to be more competitive. Secondly, there is resistance to change when an organization moves towards adopting E-business (Parreiras. F, n. d. ). In a research done by Ahmed. Z and et al. (2006), it was determined that the resistance to change will cripple the organization. Thus, it is important for the entrepreneurs to adapt to rigid cultures. ECONOMIC CHALLENGES In the aspect of economy, the nation’s currency plays a vital role. Thus, it also poses as an obstacle for traditional businesses.In areas of importing and exporting, entrepreneurs need a mutually agreed upon currency (e. g. U. S dollar) due to different payment methods. Furthermore, being sensitive towards exchange rates of currencies is vital as it will affect the buying decisions (Kavas. F, 2011). As compared to E-business transactions, the fluctuation in currency is harder to depict; thus, becoming a challenge for traditional businesses. On the other hand, E-businesses also experiences economic challenges. Firstly, the free entry into the digital market will be a problem for entrepreneurs.As there are minimal barriers to entry, it would mean that E-businesses are operating in a highly competitive market whereby competitive advantage is almost impossible as highlighted by M. Hassan & E. Harris (2007). With this tight competition among entrepreneurs the issue of imitation of products will arise. As described in the related research paper, it was revealed that a massive amount of imitation took place as it was a cost effective method to most entrepreneurs. Therefore, it becomes an obstacle for E-business entrepreneurs. COMPETITIONWhen it comes to t raditional businesses, entrepreneurs will definitely face competition. One major factor that entrepreneurs have to consider while exporting their products is the difference in time zones. An article written by Henricks. M (2006) explains that the time zone differences will cause a lot of difficulty in terms of decision making, planning shipment, organizing logistics and more. As opposed to E-business, E-business have online softwares that enables smooth communication through digital platforms regardless of time zone differences.For E-business entrepreneurs, the first challenge here is the difficulty to obtain capital large enough for any ventures. This includes the research and development needed for the product. In the report â€Å"Managing Worldwide operations & Communications with Information Technology† (2007), it was highlighted that many venture capitalist will incur higher risk with large sums of capital. This is because there is high uncertainty of success for entrepr eneurs to compete in an open market environment. In addition, another factor arises which is also known to be labour market exuberance.This is described as an irrational competition that arose with the increasing need of technical skills (e. g. software programming). Therefore, firms will likely experience a short supply of skilled workers (Wright. P & Lee. D , 2000). POLITICAL & LEGAL CHALLENGES In the area of political and legal, there is a major concern of the country’s policy for traditional businesses. Entrepreneurs need to abide by both the local and the other country’s laws and regulations while selecting to export goods over. This is due to several laws are made according to the country’s culture and beliefs (Kavas.F, 2011). For instance, products that contain pork are restricted in Islamic countries due to their religious beliefs. Apart from that, there is an essential concern of the security and privacy of E-businesses (Parreiras. F, n. d. ). A researc h on â€Å"Security and Trust in E-Business† by Valmurugan,M. S (2009) discussed the unawareness of E-business transactions and the degree of confidentiality of E-business transactions. Without the trust of consumers, entrepreneurs in the E-business line will be facing serious competition to obtain their share of the market.Another major concern is the government regulations. The government needs to play a role to protect the consumers against unfair and deceptive trading especially when it comes to Internet banking (Kay. A, Hafeez. K & Siddiqi. J, n. d. ). Therefore, this would become a small obstacle for E-business entrepreneurs as they need to adhere closely to government policies. CONCLUSION This research paper covered numeral aspects of traditional businesses and E-businesses with respect to the macro-environmental challenges faced by the entrepreneurs.Because we live in a rapid changing environment, businesses will continuously face multiple challenges. The traditional way of business may have been successful in the past, but now is the time for change. E-business enables organizations to reach global markets; thus, crossing borders with less restraint from trade barriers. Evidently illustrated by Fleenor. C & Raven. P (n. d. ), the adoption rate of internet is growing tremendously and that governments of international countries recognises such growth; hence, promoting it as well within their country (e. . E-government). Although there may be areas that E-business is a challenging area to pursue; however, without such obstacles businesses will never revolutionalize. Therefore, it goes to show how businesses have evolved over the decades. Business entrepreneurs need to be more innovative as traditional business will soon be uncompetitive. 1,111 words Reference List Ahmed, Z. et al. (2006)  RESISTANCE TO CHANGE AND ERP IMPLEMENTATION SUCCESS: THE MODERATING ROLE OF CHANGE MANAGEMENT INITIATIVES. [online] Available at: http://web. usm. my/aamj/11. . 2006/AAMJ%2011-2-1. pdf [Accessed: 26/9/2012]. Fleenor,, C. and Raven, P. (n. d. )  Barriers To Effective E-Business In Developing Countries. [online] Available at: http://www. google. com. my/url? sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&ved=0CC0QFjAB&url=http%3A%2F%2Fcluteonline. com%2Fjournals%2Findex. php%2FIBER%2Farticle%2Fdownload%2F3912%2F3957&ei=dTRlULXHAcnlrAeTuICgDg&usg=AFQjCNFo3MqMmHHbqbtHGpyGH9MPiLUQhA&sig2=NnGIv4mCofIkSx211W9PVw [Accessed: 28/9/2012]. Hassan, M. and Harris, E. 2009)  Entrepreneurship and innovation in e-commerce  . [online] Available at: http://www. journalamme. org/papers_vol32_1/32114. pdf [Accessed: 25/9/2012]. Heerwagen, J. et al. (2010)  The Changing Nature of Organizations, Work, and Workplace. [online] Available at: http://www. wbdg. org/resources/chngorgwork. php [Accessed: 26/9/2012]. HENRICKS,M . (2006)  How Time Zones Affect Global Businesses. [online] Available at: http://www. entrepreneur. com/article/160228 [Accessed: 28/9/ 2012]. Kay, A. et al. (n. d. )  AN EMPIRICAL STUDY OF THE KEY DRIVERS AND

How Socioeconomic Status of Parents Affects Kids

How socioeconomic status of Parents affects their Children’s Development in Academics It is obvious that most people have set beliefs on when they see a family of a low socioeconomic class that their children will grow up to be the same as their parents. People believe that they will not be as likely to do well in school or even in the real world. Many psychologists have done studies that have proven that this assumption is right for the most part. Children that have parents, family and neighbors of lower socioeconomic status tend to not do as well in school as their peers of a middle or upper socioeconomic status. Duncan, Kato, Brooks-Gunn & Klebanov, 1993) (Duncan, Kato, Brooks-Gunn & Klebanov 1993) conducted a study to determine whether a child’s socioeconomic status had any correlation with their academic development, ethnicity and if they were raised by a single parent. (Duncan, Kato, Brooks-Gunn & Klebanov 1994) They hypothesized that children of a lower socioecon omic, status and neighborhood would have a direct relation to lower IQ of the children they measured at age 5. They measured each child in their study at age 5 from all of the different socioeconomic, ethnic, and parental backgrounds. They found a strong correlation of a person’s economic status and economic status of the people around them to their IQ. (Barry 2005) also did a study that involved whether or not socioeconomic status had any relevance on whether a child would have better or worse test scores in 10th grade on a standardized test based on the child’s economic status. He hypothesized that children of a lower economic status or of a Hispanic, African American, or Indian will tend to have lower Scores than children of white children with a higher socioeconomic status. His results show that the strongest predictor of student test scores is socioeconomic status. (Barry 2005) He states that ethnicity combined with economic status plays a large factor in how well the students did on the SAT standardized test. For example, in 1991-1992 African American students placed significantly lower on the SAT than White students. (Barry 2005) Janet Currie and Joshua Goodman have also done a study in that they were looking for a correlation between socioeconomic status of a child and how well they would perform on certain standardized tests. Their results have shown the same positive correlation as in the other two articles. (Investments in education pay off in the form of higher future earnings, and differences in educational attainments explain a significant fraction of the adult variation in wages, incomes, and other outcomes. But what determines a child’s educational success? Most studies point to family background as the primary factor. But why does background matter? While many aspects are no doubt important, research increasingly implicates health as a potentially major factor. The importance of health for education and earnings suggests that if family background affects child health, then poor child health may in turn affect education and future economic status. ) (Currie, Goodman) After reviewing both ideas of ethnicity and socioeconomic status having or not having a measurable outcome on academic proficiency, psychologists are able to determine that while not 100% of lower economic status students and ethnic students performed worse an overwhelming majority didn’t perform as well as their upper economic status or white peers. References Barry (1994) The effect of socioeconomic status of academic Acheviement Wichita State University, Thesis Paper Duncan, Kato, Brooks-Gunn & Klebanov (1993) Economic deprivation and Early childhood development Currie, Goodman (ND) Parental socioeconomic status, Child Health, and Human Capital