Patroklos is not very important to the Iliada Essay Example for Free

Patroklos is not very important to the Iliada Essay Patroklos is introduced as the companion of Achilleus – the great warrior – which automatically makes him significant to the poem as a whole. His relationship with Achilleus is first shown in book 9 where Patroklos obeys everything Achilleus asks him to do and is names as ‘his companion Patroklos’. This shows they have a good relationship which is important as it shows the audience another side of Achilleus, one where he is loving and caring and human enough to crave the company of a close friend. However the menial tasks Patroklos is first asked to perform could have been undertaken by any common slave and therefore in this scene he does not come across as particularly important to the poem. This close relationship is emphasized a number of times throughout the poem, in particular when Achilleus ‘gave a silent signal to Patroklos with his eyebrows’ which would indicate that their relationship is such that Patroklos understands what Achilleus wants him to do through facial expression alone. Moreover, in book 11, Patroklos is referred to as the ‘pleasure of my heart’ by Achilleus, suggesting that their relationship is deeper than just friends. Patroklos’s role in Achilleus’s life is explained in part by Nestor who speaks of the advice given to Patroklos by his father ‘by birth Achilleus is superior to you, but you are the older. He is far stronger than you, but your proper task is to give him words of wisdom and advise him and guide him – and he will listen to you for the best. ’ This indicates that Patroklos has throughout his life influenced Achilleus and continues to do so, meaning he is crucial in understanding Achilleus’s character. Nearing the end of book 11, Patroklos cuts an arrow from Eurypylos thigh and stops the bleeding which shows he is compassionate and endears him to the audience which may be a device employed by Homer to emphasize the sadness of Patroklos’s death later in the poem. This scene is also used to show the brutal effects of war due to its gory nature. After book 16 Patroklos comes to the forefront of the poem. Achilleus instructs him to don his armour and push the battle from their ships. Achilleus once again shows his feelings for Patroklos as he prays to Zeus that he will come back safely. Zeus does not grant this, foretelling Patroklos’s death. During this book, Patroklos has his aristeia which adds excitement and impact to the poem. Patroklos’s fighting lasts for many pages and is a significant part of the poem, showing his importance overall. Patroklos’s fight with Sarpedon is important as Zeus becomes upset over the death of his son and although he allows the Achaians to take his armour, he ensures the body is well looked after – showing a loving side of Zeus that is not often seen during the poem. Without Patroklos this scene would not have been able to take place. Patroklos’s death is told over many lines and begins with Euphorbus stabbing him in the back as he is too scared to face him in battle. This shows once again how brave and important Patroklos is. The passage describing Hektor killing Patroklos is very descriptive and includes mockery from Hektor and Patroklos’s final speech. This once more makes the audience feel for Patroklos as he is cruelly mocked in the last moments of his life. The next book is entirely dedicated to the fight over Patroklos’s body which indicates that he was important and a large part of the poem. There are also many men willing to defend Patroklos which shows he was well loved ‘I would gladly stand by Patroklos and defend him, since his death has touched right to my heart’. At the beginning of book 18 Achilleus learns of the death of Patroklos ‘and the black cloud of sorrow enveloped Achilleus†¦he lay there with his whole body sprawling in the dust, huge and hugely fallen, tearing at his hair and defiling it with his own hands’, this passage shows how much Achilleus cared for his friend, he is so overwhelmed with sorrow that he is unable to speak. Even the serving women ‘shrieked loud in their hearts’ grief’, enabling the audience to understand that he was loved by all, emphasizing the pain of his death. Furthermore, Patroklos’s death makes Achilleus feel guilty ‘let me die directly, since I was not able to help my friend at his killing’ and spurs him to fight once more. The sensitive side of Achilleus is bought out again during this ‘his warm tears falling when he saw his trusted friend lying there’. Patroklos’s death is the catalyst needed to make Achilleus rekindle his fighting spirit ‘but now Patroklos†¦I shall not give you burial until I have bought here†¦the head of Hektor. ’ Which shows that without Patroklos Achilleus would not have fought again, meaning he is catalytic in this part of the Iliad. Finally it is made clear that Patroklos was instrumental in Achilleus’s attachment to Briseis. ‘You were always gentle. ’ Briseis explains that Patroklos was the one who arranged for her to marry Achilleus, and since this all began because Briseis was taken away this shows that without Patroklos’s intervention Achilleus would not have been so angry in the first place and would not have refused to fight, making the Iliad into a very different story. Therefore, Patroklos was key in shaping the poem into the story it became.

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.

Condominium Living in Malaysia

Condominium Living in Malaysia The concept of condominium living in Malaysia is already promoted for few years before and almost all the people in the city will accept this type of housing. Due to the rapid rate of urbanisation and scarce of land available, condominium become the most popular housing types which lead to maximise the usage of land available with providing the facilities. Condominium living has become more common in a feature of the residential landscape in many parts of the country, especially in densely populated areas such as Kuala Lumpur and Penang. This is mainly cause by the scarcity of land in big cities and rapid urbanisation due to massive population migration. Other than population migration, the needs of people and changing lifestyle have also contributed to the development of the high-rise building over the country. Limited land has driven up the prices of properties, especially the place such as those in the Kuala Lumpur city centre and the fast developing corridors in the Klang Valleys (The Star, 2004). Since Malaysia is now promoting a policy, housing-owning democracy with a target which every family will own a house. With this policy, there is an opportunity to let the people choose what kind of housing is likely to be bought. Condominium living can open up an opportunity to strengthen the relationship between three major ethnic for racial integration. It is to help the political and economic stability in the country. All the residents will share their responsibility and give their effort over the common areas and facilities such as corridor or the recreational facilities within the condominium. There are many reasons why people make their homes in condominiums. Some is because their children have grown up and moved out, and their homes are now too large. Others consider it because it is cheaper to buy a unit in a condo rather than a landed property in the location they want (National House Buyers Association, 2009). Living within the condominium where neighbours can be easily call upon when problems arise will increase the mutual understanding between the neighbours. Condominium living is about lifestyle and buying a condominium is actually investing in a lifestyle (Chan, C. K., 1991). Some rich people will buy the condominium just because of the facilities it is provided. They can just rent the condominium in term of it location or equipped by good facilities such as 24-hour security, swimming pools and barbecue pits. From this chapter, we will understand concept of condominium development, definition of condominium, the types of condominium, basic instrument for purchasing condominium, the units, common areas, Strata Title Act (1985) and Building and Common Property (Maintenance and Management) Act 2007 (Act 663). The Concept of Condominium Development: Condominium means to control (dominion) a certain property jointly with (con) one or more persons (Chan, C. K., 1991). This housing development is a form of co-ownership over a multiple-unit property, for instance flat. The co-ownership will own a unit and have their own interest in the air space and an undivided interest in the common area, such as corridor, lobbies, playground, garden, swimming pools and car park. Condominium development is to be introduced and solved the problem because of the rapid urbanisation and limited of land available. Due to these problems, condominium is more popular in the crowded city such as Kuala Lumpur. Desa Kudalan project was the first project launched in Kuala Lumpur which was located in the up-market area of Jalan Pekeliling (Chan, C. K., 1991). Demand of condominium is rise in crowded cities and there will be some of the factors that contributed to the demand such as scarcity of land and high land cost. Because limited land available in cities, it driven up the land price goes up. Condominium developments help the citizen to have their own house which in the multiple-unit property, condominium where each individual has their own interest known as unit. As the population rise, some cities are facing shortage of housing. Multiple-unit property will help the citizen to have their own house which fulfilled the housing democracy which now promoting by the government where every citizen owns their house. People nowadays are more educated and they will meet and satisfy their need to fulfil the life they want to be. Changing lifestyle is one of the factors for the contribution of condominium development. There are many facilities provided by living in condominium such as24-hour security and swimming pool. The types of condominium development are different in the form of low rise and high rise, integrated linked type, cluster of repetitive nature, in varying community density, in more central urban region and the suburban as well as areas of resort, in varying cost values; but in all cases, the concept of mutual benefitting community living remains the basic (Sze To, K. Y., 1979). The concept of condominium development is consider quite success in the crowded city compare to rural area. This has helped to create a market for condominium housing and citizens have more choice to choose according to their favourite. Definition of Condominium: Con-dominium is derive from two word which mean joint sovereignty or in the more historical sense joint control of a State by other States. In housing development, it is referred to subsidiary title coupled with common title or as defined under the National Land Code in previous days (Sze To, K. Y., 1979). The word condo or condominium comes from the two Latin words- con means together and dominium, means property. A condominium can be an apartment, house, townhouse or a unit in an apartment house in which the units are individually owned. Hence, there is always common property owned with others- recreation areas, lawns, basement, garage as well as the individual units are owned outright ( Kalia, A., 2007). Others than that, condominium is the home ownership that owned by the individual units, which is known as condominium units. Undivided interest means a share or right that owned by the unit holder, but the ratio or the share cannot be defined apart from the whole (Holeman, J. R., 1980). Each unit owner has the same interest which is called shared rights towards the common areas that used together, for instance swimming pool, car park, barbecue pits, elevator and others. Management of the condominium is responsibility to well manage the common area in order to provide a harmonies environment within the condominium. An amount of money, which is management fee, will be paid by the unit owners every month in order the property management are sufficient money to manage the common areas. Normally, home owners will form an association, to maintain and manage the common areas. There are various types of design unit within a condominium, such as studio unit, penthouse and duplex. Penthouse and duplex are expensive condominium living and normally rich people will purchase this kind of condominium to match their luxury lifestyle. Studio unit is consists of dining, living and bedroom that combined together to become a large room. The kitchen facilities as a part of the central room. Bathroom in this unit has its own smaller partition. Penthouse which high classification in the condominium is located at the top of the building. It is separate from with others building to have privacy by unique design such as high ceiling. Duplex consist of two floors connected by internal staircase in condominium. Types of Condominium: There are several type of condominium such as residential condominium, non-residential condominium, standard condominium and phase condominium. Residential condominium is owned by the individual units which the owner will occupy for living purpose. Some of the owner will rent out to others to earn the rental income. They will pay monthly maintenance fee to management department of condominium for maintaining and managing the common area that all the owners have the shared right on it. There is also non-residential condominium can be found in the property market such as hotel, services apartment, retail shop and office building. The structure is the same with the residential condominium but the difference is the usage of the building. Standard condominium is just a general type of condominium that can be found in any country. This kind of condominium is subdivided into units and common area. Hence, this kind of owner will own different but inseparable entities. One is the well defined space that used for residential purpose; one is the common area which the owner have shared interest on it. This shared interest between the owners cannot be defined from the whole part. Phased condominium is a condominium developed according to stages. Normally, the time to complete the development will not exceed 10 years. Hence, the condominium development size will increase from time to time until the development is complete. The advantage of this type of condominium is the purchasers do not have to wait for so long for the development to complete. There is also a benefit for the developer which the income does not influence by the period to occupy or awaiting title. This type of condominium will use phase one to be the name that completed first. The owners is also own different but inseparable entities. Basic Instrument for Purchasing Condominium: Before the condominium can be purchased, there are three instruments used in order to purchase condominium, deed of Mutual Covenant, declaration of condominium and bylaw of condominium association. Deed of Mutual Covenant: When a person purchases a property where there are common areas that need all the involvement of all the unit owners, the owner will asked to enter into a Deed of Mutual Covenant (DMC) while the Sale and Purchase Agreement (SPA) is signed upon. Deed of Mutual Covenant is an agreement of mutual involved between the developer and the purchaser. The contents different from developer to developer, and they are distinctly separate from those in the SPA (National House Buyers Association, 2004). Housing Development Act does not regulate the contents in DMC compare to Sale and Purchase Agreement. The contents consist of the clauses which assure the purchaser and the developer. There are some clauses are as follows: Maintenance Charges. The maintenance charges are the agreed amount that valid for a period. After the period, the agreed amount will be increased to a reasonable amount. Undertaking Not To Lodge A caveat. DMC restrict the purchaser or owner to lodge any caveat on the property. Right of the developer to cease supply of utilities. Developer has the right to cut the utilities such as electricity and water supply, in case of any default or delay payment by the resident. Consent fees. National House Buyers Association (2004:1) stated. The amendment to the Housing Regulations, effective Dec 1, 2002, developers can only charge a sum of 0.5% of the purchase price or RM500, whichever is lower. This is regardless of when the SPA was signed. The amended Housing Regulations also states that: No housing developer shall collect any fee by whatever name called for giving his consent to any purchaser or subsequent purchaser of a housing accommodation to assign his rights and benefits to and in the contract of sale to any financial institution providing a loan for such purchaser to finance or part finance the purchase of the housing accommodation Period of validity of the DMC. The validity period of DMC is the strata titles have been issued to the each unit owners and the management corporation is formed.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  There are many dispute causes by the clauses between developers and buyers. The maintenance charges will be increase to a reasonable amount after certain period. Some buyers do not check during signing the DMC or do not understand what is the reasonable amount will be stated. The disputes begin with developers after certain period when buyers realise they have to pay more for maintenance charges. Although the buyer is owned the property, he is no right to enter any caveat to his own property to secure his rights. Government should solve the dispute between developers and buyers by standardize contents of DMC like Sale and Purchase Agreement to ensure the equilibrium balance between developers and buyers. Declaration of Condominium:   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  The declaration of condominium (also known as the declaration of restrictions or master deed) establishes the definition of the private and common elements within the condominium community, and outlines the rights and obligations of the owners. It is actually a legal instrument recorded that create a condominium development under law. This declaration of condominium will set up a fund to maintain the property, and creates the condominium association. Instructions for how amendments are to be when the change of conditions of demand should be given by the declaration of condominium. For instance, the developer has to prepare a declaration of condominium to fully state the ownership rights of the owners according to their units (Holeman, J. R., 1980). Articles of Incorporation:   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Constitution condominium community is a set of rule and regulation which same as the articles of incorporation and declaration of condominium. The articles of incorporation will constitute the rule and regulation for the purpose of condominium and it will be stated there whether it is for investment purpose or just for resident occupation. The condominium will operate according to the rules and regulation when the article of incorporation is legally bound while condominium by-law will establish the guidelines of condominium. Membership requirements, election procedures, and the powers and duties of the directors and officers are regulated by by-law. They determine how the finances are to be handled, and how the project is to be maintained, insured, and restricted. By-law will continuous change and amend to meet the suitable condition (Holeman, J. R., 1980). Bylaw of Condominium Association; By-law should be abided by the condominium unit owners. Developers or the purchaser of the units are generally draft the rules and regulation of condominium in by-law of condominium. Normally, by-law will establish the electing officers or board members procedures of the condominium association, inform the member when the meeting is being held, and also responsible for the building maintenance and insurance in common areas. Some of the restrictions may impose to restrict unit holders to do something against the rule and regulation or penalties for not compliance to the rules (Answer Corporation, 2009). Rules and Regulation; Rules and regulations are required by managing the condominium as the by-law gives the rules for living together with harmonies. These rules and regulations will determine and enforce by the board of Director. Some of the rules and regulations are stated as below: Homeowners Dues. The monthly homeowners due must be paid according to the period that has been determined. Condominium may take some action towards homeowners who are refused to pay it. Common Elements. The homeowner or residents are not allowed to making loud noise which will disturb other residents within the period that has been determined. The balconies must be neat and clean in appearance. No any blocking items or store room are allowed to be place at common area. Parking. Vehicles have to be parked in the designated parking spaces. Vehicles are not allowed to park their vehicle at the prohibited area such as in front of garage. Pets. Some of the condominium may allow the residents to have their pets. Some condominium will not. If allowed, it must consistent with the responsibilities associated with living in a condominium. The Unit: Unit is the description which usually contained in the declaration of condominium. The most important element is boundary described, because homeowners are responsible to maintain, manage, insure and decorate their own unit. Each unit owner shall be entitled to exclusive possession of his apartment, subject to the provisions of the condominium documents (Holeman, J. R., 1980). Common Areas: The documents refer to common area shared by condominium owners as that part of the projects exclusive of all private units. The ownership of each condominium unit includes an undivided interest in, as well as rights and title to these common areas (Holeman, J. R., 1980). The common areas are divided into two: general common interest and limited common elements. Generally, common areas include the land, main walls, foundations, roofs, hallways, elevators, swimming pools and other recreational areas and parking lots. Detached carports and garages are sometimes owned by the association and assigned to individual unit owners, and sometimes they are owned with the unit (King, B., 2003). Limited common elements are a physical part of the common areas, but are for the exclusive use of a particular unit owner or group of owners. Limited areas usually refer to parking spaces, patio space, Storage space, and entrances ways (Holeman, J. R., 1980). Neighbourhood Link (2009:1) explained. Limited common elements could also be any air conditioning or heating units, chute, flue, duct, wire, conduit, bearing wall, bearing column and other fixture, whether located within or outside of the boundaries of a unit, which serve only that unit or are allocated solely to that unit. Any shutters, awnings, window boxes, doorsteps, stoops, porches, decks, balconies, entryways, patios, exterior doors and windows, other fixtures designed to serve a single unit, but located outside the Units boundaries are limited common elements allocated exclusively to that unit. Strata Title Act (1985): The interest of unit holders and developers are subjected in sub-division of building for subsidiary titles (Strata Titles). This legally abided title has contributed a more effective implementation when some of the disputes are arise (Kok, S. T., 1979). The Federal and Director Generals Land and Mines Department (2009:1) explained that strata Title was first introduced in 1966 by the Malaysian National Land Code 1965, to better cope with the legal ownership of multi-storey buildings. Previously, it was known as subsidiary titles by reference to the buildings erected on alienated land. By 2007 strata title legislative amendment, the strata titles concept was extended to be applied for land parcels created on alienated land. Definition: Strata title is a form of ownership for multi-storey buildings or blocks or multi layer of land on a piece of alienated land. The Strata part term refers to subdivided units being on different levels (The Federal and Director Generals Land and Mines Department, 2009). Management Corporation is formed under Strata Title Schemes where combination the individual parcels and common property with a self-governance of Strata Corporation. The individual parcel is indicating condominium, apartment, town house, and each of the individual units will be hold a different title. Management Corporation is formed automatically when the strata title is registered by opening of a Book of Strata Register (The Federal and Director Generals Land and Mines Department, 2009). The Management Corporation has to elect a council who responsible to perform the MCs duties and powers and carry out the MCs business. The council is consisting of not less than three and not more than fourteen proprietors of multi-storey building. Under section 41 of Strata Title Act 1985, the first Annual General Meeting (AGM) must be conducted within one month after expiration of the initial period. Initial Period is the period commence from the day of the MC is formed and the end on the day on which there are at least one- quarter of aggregate share units, not including the proprietor of the lot who known as original proprietor of Master Title (Tan, R., 2007). By-Laws: By-laws under the Third Schedule of Strata Titles Act, 1985 are applicable to all subdivided buildings. Additional by-laws and continuous amendment can be made by management corporation so that by-laws does not contracting with the Third Schedule of the Strata Titles Act, 1985. All owners in Management Corporation must comply the additional by-laws as long as the law is applicable. And the additional by-law shall be publishing in the public for their awareness. Records of all such by-laws must be properly kept by Management Corporation for inspection (National House Buyers Association, 2009). Requirement of Subdivision of Building: Homeowner will be given a separate title which is sub-divided any building into each individual title of the proprietor of the land with approval of The Federal and Director Generals Land and Mines Department. Any building which intends to sub-divide to an individual title has to be subject to some of the conditions as follow: The land is only held as one lot under registry title. Building must have 2 or more storeys above the ground level, with an area at least 5,000 square feet. The building must approved and certified by a Licensed Surveyor. The boundary of the lot with will be examined carefully by him. The sub-division does not contravene any restriction in interest in the land or any requirement that has been gazetted in title. The building has to be constructed in accordance with the plan submitted where the planning permission was required by for the building and it should certified by Architect(register under the Architect Ordinance 1951). here is no item of land revenue is outstanding in the respected land. Formation of Management Corporation: MC is the medium through which the proprietors control and manage the strata scheme pursuant to the Strata Titles Act 1985 and the Rules made there under. If it is a subdivided building or multi-storey building, it is automatically register under strata title. The MC is known by the name appearing in the book of the strata register relating to the subdivided building (National House Buyers Association, 2009). It is a body corporate having perpetual succession and a common seal. It is not necessary to register it under the Societies Act 1966 or any other law. In the case of a subdivided low-cost building, however, the MC does not come into existence automatically with the opening of the strata register. Under section 64 of the Strata Titles Act 1985, the MC comes into existence upon the completion of the transfer of strata title in respect of all the parcels by the original proprietor. On the other hand, the proprietors, other than the original proprietor, having share units totalling more than half of the aggregate share units of all the parcels may apply o the Director of Lands and Mines for an order to establish the management corporation as provided for under section 64A. Duties, Powers and Responsibilities of the Management Corporation: Normally, the duties, powers and responsibilities of MC are under section 43 of the Strata Title Act, 1985. National House Buyers Association (2009) stated that the MC should exercise their duties, powers and responsibility according as below: To collect the contributions from parcel proprietors. To purchase the movable property for the use as common property. To secure the repayment and the payment interest. To do all things necessary for the performance of its duties. To manage and maintain the common property. To insure and keep insured the subdivided building against fire and other risks. To pay insurance premiums, quit rent and other rates. To comply with any notices or orders given by an public authorities to execute any works in respect of the reasonable time. To prepare and maintain a strata roll for the subdivided building. To be the custodian of the issue of the document of title of the relevant land. Tot purchase additional land, grant or accept an easement. To provide an appropriate receptacle for postal deliveries. Financial Management: Management Corporation has to establish a management fund in order to manage and maintain, control the common properties, paying taxes, rates and insurance. Some acquisition and investment activities may also allowed for MC to proceed. MC will decide the amount of homeowner to levy the contribution according to their respective share unit and the interest rate whoever homeowner is in respect of late payment of contribution for maintain the management find accounts. A portion of money from the management fund account are distributed for painting the building, purchasing movable property for the purpose of common property, replacement of fixture and fitting in common property and others expenditure for maintenance. Building and Common Property (Maintenance and Management) Act 2007 (Act 663): Building and Common Property (Maintenance and Management) Act 2007 (Act 663) was first came to enforce on 12 April 2007. It is an Act to provide for the proper maintenance and, management of buildings and common property, and for matters incidental thereto (Lee, S. S., 2007). There are some problems during the duration after vacant possession is delivered by developer to purchaser and before the establishment of the Management Corporation. Hence, this present law is to cover what is inadequate in addressing the problems during that period. The developer is responsible for the maintenance and management of the common property before the formation of the Management Corporation (Lee, S. S., 2007). The maintenance and management of building and common property under the Building and Common Property Act 2007 covers all types of building that will be or have been subdivided and issued with strata titles namely residential buildings such as condominiums flats, apartments and gated community developments, commercial buildings such as office blocks, shopping complexes, service apartments, mixed developments and industrial buildings. In respect of building with strata titles, it includes all parcels, accessory parcels and common property (Maidin, A. J., 2007). According to the section 4 of the Building and Common Property (Maintenance and Management) Act 2007 (Act 663), a Joint Management Body is formed which comprise the developer and purchasers to administer the maintenance and management of the building during the initial period before the Management Corporation fully takes control of the common property of the development from the developer (Soong, D., 2007). Joint Management Body: The first meeting of the Body has to be convoked by developers no later than 12 months from the commencement of the act for the building concerned was completed before the commencement of the Act. In cases where the building is completed on or after the commencement of the Act, the meeting must be held no later than 12 months from the delivery of vacant possession to the purchasers. Unlike the Management Corporation, the developers duty to convene the first meeting of the Body is effectively tied to the delivery of vacant possession to the purchasers and not effected by how long it takes the developer to apply for and procure the opening of the strata register for the development or whether the developer has sold and transferred 25% of the development to purchasers (Soong, D., 2007). Formation of Joint Management Body: Before the JMB is established, the developer has responsibility to carry out the maintenance works and to ensure the building is free others risks. Developer has the duty to have a first meeting with all the purchasers within specified period. If developer fails to do so, the developer will be fined not more than RM25, 000 or imprisonment in a period not more than three months or both. Duties of Joint Management Body Joint Management Body is a body corporate, hence there are boned to be sued if anything found in default. The duties of the JMB are, among others, to: Maintain the common property and keep it in good serviceable repair. Fix and impose charges for the maintenance works. Insure the building and apply insurance moneys received for rebuilding and reinstatement. Prepare and maintain a register of all purchasers. Ensure that the Building Maintenance Fund (BMF) is audited and provide financial statements to purchasers. Enforce house rules (Wong, A. F. H., 2009). Powers of Joint Management Body: Joint Management Bodys powers under Building and Common Property (Maintenance and Management) Act 2007 (Act 663) are almost the same with those of the Management Corporation under Strata Titles Act. The Body is essentially a form of Pre-management Corporation (Soong, D., 2007). In essence, the Body is required to perform all acts required for the proper maintenance and management of the building, such as maintaining audited accounts and enforcing house rules for the proper maintenance and management of the building. The JMB is empowered to: i. Collect maintenance charges from purchasers. ii. Authorise expenditure for carrying out the maintenance works. iii. Recover monies due from purchasers. iv. Acquire property for use by purchasers in connection with the common property. v. Secure the services of a person to undertake the maintenance works. vi. Make house rules (Wong, A. F. H., 2009). Building Maintenance Account: Financial statements which include the maintenance and management of the development are specified by the Act has to be maintained by developer and the Body before the account is being passed to the Management Corporation. Ensuring funds to be audited which held for the maintenance of the development and provide the audited financial statements for the information to purchasers is part of the duty of the Body (Soong, D., 2007). In each development, developer is required to open a Building Maintenance Account before vacant possession is delivering to homeowner. Other than that, developer also required to deposit all the money received for the purpose of Building Maintenance Account which the money does not go directly to developer. Building Maintenance Account must to be audited by a public and professional auditor, and the auditors report must be attach and report to Commissioner within 14 days. Commissioner has the power to engage their own auditor to investigate the entire document related, such as account of the developers company. Under Part IV Section 16, before delivery of vacant possession, the developer of a strata development must open a Building Maintenance Account (BMA) in the name of the strata development for each development. This applies to all newly completed strata development where the vacant possession has not been delivered to the purchasers as of 12th April, 2007. For developments where vacant possession has been given but the management corpora

The Hutu Tribe :: essays research papers

The Hutu Tribe   Ã‚  Ã‚  Ã‚  Ã‚  The culture of the Hutu and Tutsi tribes of Rwanda, Africa interests me for many reasons. One reason is that they are so diverse from our American way of life. Another reason is that I have heard a little bit about them in the news and by talking to people. This sparked my interest and made me want to learn more about them. I will cover a wide variety of information in my report. This will include the land where they live, their way of life, their history and ancestry, and what the government is like in Rwanda. I don't expect the reader to become an expert on the Hutus and the Tutsis, but I do hope that the reader gets a general understanding about what is going on in their section of Africa. I have learned lots of new information from doing this report, not only about the Hutus' and the Tutsis' culture, but about the whole continent of Africa.   Ã‚  Ã‚  Ã‚  Ã‚  The first topic that I will cover is about the people that make up the tribes of the Hutu and Tutsi. The total population of the country of Rwanda, where most of their events have taken place is 7,800,000. The population of the Hutus is 3,000,000 and the Tutsis number only 1,000,000. The physical traits of the Hutus and Tutsis are very different from each other. The Hutu have larger noses and larger all around facial features than the Tutsis. Most Tutsis are seldom less than six feet tall, while the Hutu are very short people. The Tutsis are related to the Masai and the people along the Nile, while the Hutus have a Buntu history. As you can see the people that make up the tribes of the Hutus and the Tutsis are a very diverse group of people.   Ã‚  Ã‚  Ã‚  Ã‚  The way of life for the Hutu and the Tutsis is considerably different. Traditionally, the Tutsi were the wealthy, upper class and the Hutu were little more than slaves. Now the power is a little more equally divided, but is still more in favor of the Tutsis. The Hutu are mainly peasant farmers, while the Tutsis are mainly animal stock breeders. One thing that the Hutu and the Tutsi have in common is that they are both a Buntu language speaking group. The basis of the natives' religion is a belief that there is a supreme principle of good known as Imana. Followers of this religion use magical amulets in their rituals. One half of the people in Rwanda are Christian, and most of the Christians are

What Caused The Downfall of Sparta? :: Ancient Greece Greek History

What Caused The Downfall of Sparta? Hypothesis: Sparta collapsed because they did not allow the helots to fight in battle The Beginning of Sparta In about 100 BCE, the Dorians invaded Greece from the North. During the Dark Ages, the Dorians made their way south, capturing the inhabitants of the lands they passed through as helots. At the beginning of the Dark Ages, it is thought that there were many Dorian settlements in Laconia, each with their own helot population. At some time during the Dark Ages, Sparta overtook these fellow Dorian settlements and their helot populations, as well as control of the whole of Laconia. The Spartans kept the helots as a huge, strong slave race and, although they did not enslave their fellow Dorians, the other Dorians were made perioci, meaning "those who live round about". The perioci were needed to be the craftsmen, tradesmen and manufacturers for the Spartans, who were trained as full time soldiers. At the end of the Dark Ages, there was nothing exceptional about Sparta (except her control of the helot population) but from about the middle of the 6th Century BCE, Sparta gradually turned away from the rest of Greece. They no longer welcomed visitors, cut their trade ties, stopped building ships and when the rest of Greece began using coins instead of iron spits, Sparta continued to use the spits. Sparta still had poetry and music, but instead of listening to new poems and songs, they learned only the compositions of the past, and new poets and musicians were not welcomed. Sparta still produced pottery and metal work for every-day use, but it was of poorer quality than the work of other cities. Spartans no longer participated in athletic festivals in other parts of Greece and the whole city became secretive and withdrawn, refusing to communicate with the rest of Greece. Education The Spartans were raised and educated to be perfectly obedient and obey the state without question. Spartan education had no interest with literature, intellectual or academic activities and did Spartans were not taught subjects like mathematics, science or geography. Even as babies, Spartiates were treated harshly - they were made to eat whatever food they were given, left alone, left alone in the dark, and it is probable that no attention was paid to babies when they cried. A Spartan Boy's education as a soldier began when the boy was about 7 or 8 years old. What Caused The Downfall of Sparta? :: Ancient Greece Greek History What Caused The Downfall of Sparta? Hypothesis: Sparta collapsed because they did not allow the helots to fight in battle The Beginning of Sparta In about 100 BCE, the Dorians invaded Greece from the North. During the Dark Ages, the Dorians made their way south, capturing the inhabitants of the lands they passed through as helots. At the beginning of the Dark Ages, it is thought that there were many Dorian settlements in Laconia, each with their own helot population. At some time during the Dark Ages, Sparta overtook these fellow Dorian settlements and their helot populations, as well as control of the whole of Laconia. The Spartans kept the helots as a huge, strong slave race and, although they did not enslave their fellow Dorians, the other Dorians were made perioci, meaning "those who live round about". The perioci were needed to be the craftsmen, tradesmen and manufacturers for the Spartans, who were trained as full time soldiers. At the end of the Dark Ages, there was nothing exceptional about Sparta (except her control of the helot population) but from about the middle of the 6th Century BCE, Sparta gradually turned away from the rest of Greece. They no longer welcomed visitors, cut their trade ties, stopped building ships and when the rest of Greece began using coins instead of iron spits, Sparta continued to use the spits. Sparta still had poetry and music, but instead of listening to new poems and songs, they learned only the compositions of the past, and new poets and musicians were not welcomed. Sparta still produced pottery and metal work for every-day use, but it was of poorer quality than the work of other cities. Spartans no longer participated in athletic festivals in other parts of Greece and the whole city became secretive and withdrawn, refusing to communicate with the rest of Greece. Education The Spartans were raised and educated to be perfectly obedient and obey the state without question. Spartan education had no interest with literature, intellectual or academic activities and did Spartans were not taught subjects like mathematics, science or geography. Even as babies, Spartiates were treated harshly - they were made to eat whatever food they were given, left alone, left alone in the dark, and it is probable that no attention was paid to babies when they cried. A Spartan Boy's education as a soldier began when the boy was about 7 or 8 years old.

The Complex Process of Selecting a Pair of Shoes Essay -- Research Ess

The Complex Process of Selecting a Pair of Shoes I plan to prove that the majority of people that invest money into footwear do so because of style. People usually don’t choose a pair of shoes because of comfort, color, or any other means at all. They usually base their decision of purchasing a pair of shoes on style. There are three tools that I will use during this experiment that will help me out. These tools will be comprised of surveys, interviews, and observations. Through survey’s I will have a sheet of paper with approximately eight questions on it. These questions range from the subjects personal preference when it comes to purchasing a pair of shoes. Whether they do it for comfort, style, color, work related, price, or even none of the above at all, to how many pairs of shoes that these individuals currently own. This will give me a general idea of what goes through peoples minds when they purchase a pair of shoes. Then I will use my observation skills. I plan to sit in the mall since it’s t he best public place for this experiment, and observe what brand, color, and style of footwear the general public wear. I will more than likely sit in front of Finish Line since they are one of the major footwear sales stores in the country. This will show me exactly what people choose to wear, but won’t tell me why. Since I wouldn’t know why they wear them I plan to interview a couple of volunteers to get their opinion. This plus the rest of my research will give me a good idea of what people choose to wear, and why. I’m going to pick a few individuals from the public that seem to keep â€Å"shoes† an important part of their lives. I will ask them the same questions that are on my survey plus a few more that will go int... ... and style go hand in hand. He said people prefer one brand name against another because of the way they look. On the other hand I interviewed a girl who said that she would buy any pair of shoes as long as it was cheap and looked nice. She said she had no real preference, and owned a lot of shoes. Price was her deciding factor and no matter how it looked, what the brand name was, or even the color, she wouldn’t purchase it unless the price wasn’t very high, or if it was on sale. I think my observations, surveys, and interviews prove my thesis to be correct. Even though not everybody uses style for their deciding factor, the majority of people that participated in my survey, and my interview with Rob the shoe salesman concludes that style usually is the number one factor in one way or another when a person purchases a pair of shoes.

The impenetrability of life

An Interpretation of the Morals of Heinrich von Kleist’s The Earthquake in Chile Akin to most writers of the Romantic Movement, Heinrich von Kleist eschewed the Enlightenment’s belief in reason, science and progress. He believed that life was too complex for it to be interpreted by reason and science. The impenetrability of life, particularly com/literary-analysis-skills-lesson-structure/">human nature, is the central theme of Von Kleist’s short story The Earthquake in Chile (1807). Set in the wake of a fictitious earthquake in Chile, the short story reveals how man-made norms prove to be useless in moments of disaster.Thus, it is only in times of crisis that the true nature of individuals and or institutions is bared (Allan, 108). The earthquake is traditionally used as a metaphor for massive social upheaval. The manner in which it inflicts damage – from the bottom, destroying the foundations of edifices – renders it a suitable allegory of public c ataclysm. Reactionary priests interpreted the Lisbon earthquake of 1755 as a â€Å"punishment† for the supposed sins of its populace.The French philosopher Voltaire wrote in his novella Candide (1759) that the Roman Catholic Church used the said catastrophe as an excuse to burn more alleged heretics at the stake (The Internationalist, n. pag. ). The Scottish historian, essayist and satirist Thomas Carlyle referred to the French Revolution as â€Å"(an) earthquake of Insurrection† (Carlyle, 409). In The Earthquake in Chile, an earthquake temporarily halted the implacable operation of social, civil, moral and ecclesiastical law over society (Fenves, 313).Subsequent events exposed the futility of searching for assurance in life through reliance on loosely-argued metaphysical biases such as morals (Allan, 108). In the context of the short story, morals are detrimental to human society and relationships. It merely results in prejudices that allow the powerful to subjugate t he weak. From the outset, it is already clear to the reader that patriarchal structures of authority control Santiago. Power lies primarily in the hands of the viceroy, the Archbishop and the paterfamilias.In addition, women are perceived as valued sexual possessions and individuals whose intellect and morality must be dictated by men. But, ironically, it is women who are expected to control the sexual ardor of men (Allan, 109). Under the guise of â€Å"morality,† women are supposed to exhibit behavior that will protect her from unwanted sexual advances from men. If a woman is subjected to inappropriate sexual conduct by a man, it is believed to be her fault – she is a â€Å"loose† woman who does not deserve respect from men.When, for instance, Don Asteron discovered that Jeronimo is having an illicit affair with his daughter, Josephe, he sternly warned her instead of Jeronimo to end the relationship. And when Don Asteron later found out through Josephe’s brother that she and Jeronimo continued their liaison, he had her banished to a convent. Josephe’s new â€Å"spiritual father,† the Archbishop, proved to be more unforgiving – he insisted that she be put to trial and condemned to death after she gave birth during the religious procession.Both Don Asteron and the Abbess strongly opposed the death sentence, to no avail (Allan, 110). The decision to commute Josephe’s sentence from burning at the stake to beheading resulted in â€Å"indignation (from) the matrons and maidens of Santiago† (Von Kleist, 175). Because they committed themselves to the patriarchal values of feminine innocence and impeccable chastity, they believed that no punishment could be too severe for a woman like her. Furthermore, a gory execution for Josephe would allow them to bask in the illusory sense of their own moral superiority.Her burning at the stake would affirm their belief that they were â€Å"good† women and she w as an â€Å"indecent† woman (Allan, 111). The earthquake, however, transformed Santiago into an entirely different society. The catastrophe destroyed the bastions of patriarchal authority – the cathedral, the Viceroy’s palace, the court, the house of Josephe’s father and the prison. As a result, the people finally learned to work side by side in order to survive. Differences in social class, gender and religion were disregarded to attain the common goal of survival:And, indeed, in the midst of these awful moments, in which all the earthly goods of man were destroyed and all of nature was threatened with burial, the human spirit seemed to open out like a beautiful flower. In the fields, as far as the eye could reach, people of all ranks could be seen mingled together, princes and beggars, matrons and peasant women, bureaucrats and laborers, monks and nuns. They sympathized with one another, assisted one another and cheerfully shared whatever they had been able to save to keep themselves alive, as if the universal calamity had made a single family of all who had escaped it.(180) For Jeronimo, Josephe and their son Philipp, help came in the form of Don Fernando and his wife Dona Elvira. After Josephe agreed to Don Fernando’s request that she be the wet nurse of his son Juan (Dona Elvira was badly injured in the earthquake), he welcomed her, Jeronimo and Philipp into his family. Don Fernando and Dona Elvira treated them well despite their background: Don Fernando was very grateful for this kindness (Josephe’s consent to become Juan’s wet nurse) and asked whether they did not wish to accompany him to that group of people who were just preparing a small breakfast by the fire.Josephe replied that she would accept that invitation with pleasure, and, since Jeronimo had no objection either, she followed Don Fernando to his family and was received most heartily and tenderly by his two sisters-in-law, whom she knew to be ve ry respectable young ladies. (179) Dona Elvira, to whose wounds Josephe was busily attending, had at one point – just when these stories were arriving most quickly, each interrupting the other – taken the opportunity to ask her how she had fared on that terrible day. And when, withanguished heart, Josephe recounted some of the main features of her story, she was delighted to see tears well up in that lady’s eyes; Dona Elvira seized her hand and squeezed it and gestured her to be silent. (180) Unfortunately, the rest of the town retained its bigotry. In the afternoon of the earthquake, a service was held at Santiago’s remaining cathedral. The sermon of the officiating priest likened the calamity to God’s annihilation of Sodom and Gomorrah. The earthquake, according to him, was God’s â€Å"punishment† to Santiago for Jeronimo and Josephe’s sin:He castigated it (the city) for abominations such as Sodom and Gomorrah had not known, and ascribed it only to God’s infinite forbearance that Santiago had not been totally obliterated from the face of the earth†¦he†¦digressed, with copious imprecations, to mention the two sinners themselves by name and to consign their souls to all the princes in hell. (183-184) The heady rhetoric of the sermon, along with emotional intensity generated by the devastating effects of the earthquake, resulted in violence. When a member of the congregation recognized Josephe in the service, an angry mob went after her and her companions.When the crowd dispersed, Jeronimo, Josephe, Juan and Dona Constancia (Don Fernando’s sister-in-law) lay dead. The Earthquake in Chile was Von Kleist’s explanation for his disbelief in the ability of science, reason and progress to influence life and humanity. He believed that science, reason and progress governed life and humanity through morals that were in favor of the powerful. The powerful, in turn, used these morals to exploit the weak and the helpless. Thus, when the powerful loses their ability to subjugate, they resort to diabolical means to regain this capability. In doing so, their real nature is exposed.Works Cited Allan, Sean. The Stories of Heinrich von Kleist: Fictions of Security. New York: Boydell & Brewer, 2001. Carlyle, Thomas. The French Revolution: A History. New York: Modern Library, 2002. Fenves, Peter David. Arresting Language: From Leibniz to Benjamin. Palo Alto: Stanford University Press, 2001. â€Å"Lisbon, 1755: The Earth Shook. † January 2005. The Internationalist. 17 September 2008. <http://www. internationalist. org/lisbon1755. html>. Von Kleist, Heinrich. â€Å"The Earthquake in Chile. † Great German Short Stories. Ed. Evan Bates. New York: Courier Dover Publications, 2003.