Construction Law Case Study Example | Topics and Well Written Essays - 2000 words

Construction Law - Case Study Example Partnering 1 means a relationship of open communication and close cooperation that involves both Government and Contractor personnel working together for the purpose of establishing a mutually beneficial, proactive, cooperative environment, alliances to contractual partnerships within which to achieve contract objectives and resolve issues and implementing actions as required. Partnering, as I define it, is the process of two or more entities coming together for the purpose of creating synergistic solutions to their mutual challenges. The Construction industry including both private and public sector clients, main contractors, consultants and specialists. The collective spend of the client members runs into billions of pounds and it constitutes the most influential pan-industry body in the construction industry. The building of a Partnering relationship is not straightforward and it is essential that sufficient time and resource is allocated in order to achieve the desired result. Effective relationships are the bedrock of Partnering and the Project culture needs to be one of full openness, honesty and trust in all dealings between the parties. .. The contract has been drafted as a purchase Order with separate Collaborate Construction terms. The former is only six pages long with additional appendices and is completed to fit the requirements of the particular project. In order to achieve a successful Partnering relationship all parties need to adopt a far greater degree of collaboration than would be found in conventional contracting. Partnering requires commitment throughout the business units and its suppliers and is not an easy option. Proactive attitudes and contributions will be required on Partnering from Project Initiation in order to obtain improved performance and shared benefits. The collaboration construction terms comprise some 18 pages and should not change, although some of the provisions may not apply, depending upon how the purchase Order has been completed. The contract is unique in that it can be used for the appointment of both consultants and contractors as a subcontract for appointment of sub consultants or subcontractors. There are already a growing body of evidence that lawyers are providing a different role from that of traditional 'legal technicians' and 'dispute adviser' in favour of movements towards 'assisting the delivery of successful projects'. Lawyers can genuinely 'add value' by explaining how a collaborative relationship can work within a supportive contraction framework. One simple step forward is for lawyers to at least explain the proposed form of construction contract (be it a partnering contract or otherwise) to the project team who will work under or in conjection with it. It is assumed that all parties fully understand what their legal obligations are and how their contract is indented to fit in with

Theoretical aspects of the phenomenon of child soldiers and scientific Essay

Theoretical aspects of the phenomenon of child soldiers and scientific issues related to the topic - Essay Example During colonial campaigns, armies would capture the children of prominent chieftains and raise them according to the colonial nation’s customs and laws. Nazi Germany used Hitler Youth movement to try to fight the Allied forces in the closing days of World War II. However each of these cases was isolated by time and geography. They were also exceptions to the level of armed combat which children performed. Ancient wars involved minors as charioteers and armor bearers for adult warriors. This practice can be found in the Bible. War in the middle ages was fought by strong men who had the physical strength to fight in those wars. Children were only used for support roles as they could not be participants in armed combat. The Roman army for instance required physical fitness as criteria for military service which only adult combatants could perform. Fighting with swords, spears, shields and heavy weapons required the use of adult combatants. During the middle ages, military units comprised of knights, heavy cavalry and infantry were extensively used in combat. They used armor and weapons which only adults could perform. The modern era of warfare began with the use of gunpowder. The modern nature of conflict saw the extensive use of muskets, cannons and cavalry against opponents. The weapons and training required to operate these weapons could only be performed by adult combatants. The adv ent of the industrial age also revolutionized the concept of warfare. By the twentieth century, modern armies were now using tanks, cannons, artillery, fighter jets, submarines and naval warships against each other. Again the use of children was mostly for support roles rather than combat purposes since the expertise required to train and use these weapons was only possible for adult combatants. During the middle ages, young boys were used as squires but their

Group Development Essay Example for Free

Group Development Essay The first stage is the forming stage. During this stage, things are characterized by much uncertainty. There is uncertainty as to what the purpose of the group is, what the structure of the group is, and who the leadership of the group is going to be. Members of the group will want to test the waters and try to determine how far they can go and what they can get away with during this stage of the group development process. The second stage is the storming stage. During this stage of group development, the members of the group accept the existence of the group but are against the restraints put on individual creativity. This can lead to serious problems if there is not some sort of compromise or agreement put in place on allowing individuals to be creative within the guidelines of the project. The third stage is the norming stage. During this stage, relationships among team members are becoming closer and group cohesiveness is coming together. The group identity is really becoming clear. This stage is considered complete when the structure of the group is completely solidified and the members of the group have decided on what is considered proper behavior for members of the group. In my opinion, this is one of the most critical stages in group development. The fourth stage is the performing stage. This stage of group development is really when things start to happen. Members of the team have fully accepted the structure and the team is working towards accomplishing the goals that were set forth initially. The group has gone from getting to know each other to complete understanding and working on accomplishing the  task at hand. The fifth and final stage of group development is the adjourning stage. This is only true for groups that are temporary. Permanent work groups are finished after stage four. This stage is normally when the group finalizes and wraps up the tasks that they have been asked to perform and then they go their separate ways. This stage is hopefully where the project is finalized and the group has been successful at accomplishing the goals that were initially set forth. Problem Identification There are two major problems that I see in this situation. The first problem that I see with this group is that there is going to be conflict of interests from each of the different groups. Each of the groups have different agendas as to how to fully accomplish the goals of the project. This can lead to problems or possibly a severe conflict between one or more of the groups. This differing of opinions and attitudes by the groups could also possibly lead to an overall failure to achieve any of the objectives of the group. The second major problem that I see with this project is demographic diversity. There are such wide and varied demographics in the school district that this project is going to take place. There could be conflicts that one demographic is being favored over another. There could also be problems that stem from this. If one demographic seems to think that another is being favored, then they could cause problems for the overall project and the goals of that project. Retrospective Evaluation The solution for the first problem will be the easiest of the two problems. During the formation of the development group for this project, common goals need to be decided upon by the development team. These goals need to be approved by all of the different entities that have an interest in this project succeeding. There should be one major common goal for the project and all of the groups should agree to this goal. If they cannot agree to a common goal, then there really is no need to go forward with the project. The solution for the second problem is going to be a little harder to solve. I believe that a non-interested third party group should be brought in to oversee the project. This will allow for an impartial representative to make decisions and also to ensure that no one demographic group is being favored more than another. If one groups interests are being pushed and another group see this favoritism, it could lead to problems and thus an overall failure of the project. Therefore, a third party should be placed in charge and there should be no conflicts because they should not favor any one particular group. Reflection The project is all about making things better for the kids of this school district. This could lead to better educational opportunities for all involved within the district. However, there are possible issues that need to be solved for this project to move forward and be a success. If these issues are addressed in the beginning then there should be resolutions and no problems should arise. This will allow a successful project and the enhancement of educational opportunities for the children of the school district.

Experiences of African Americans During the Civil War Period Essay Example for Free

Experiences of African Americans During the Civil War Period Essay The 1860s and 1870s were particularly trying times for African Americans. The Civil War which lasted from 1861 to 1865 saw America undergo social and political change as Americans struggled to redefine their idea of race and face the question of slavery. More importantly still were the experiences of blacks during and after the war as they fought to be accorded the same rights of life, liberty and the pursuit of happiness. In the early days of the war, the issue of slavery was avoided vehemently by Lincoln and Davis (Norton et al. 2008) despite it being an essential issue in the war between the North and South. In fact, freeing the slaves was never an agenda of the North. The North was against slavery because they perceived the South, who was pro slavery, as a threat to the North’s social and political order (Norton et al. , 2008). Consequently, being against slavery did not necessarily mean Northerners were not racist. In fact, many still saw themselves as racially superior to the blacks. Despite the apparent racial prejudice, blacks in the South still saw in the Union army their route to freedom. After Lincoln’s Emancipation Proclamation allowing blacks to serve in the Union cause, thousands of slaves, amongst them, one John Boston (Linden Pressly, n. d), fled their masters and joined the Union army in their fight against the South. Many blacks sought to assert their manhood despite discrimination in the army through the display of bravery and valor. Still more died, like the Fifty-fourth Massachusetts regiment, in their fight for equality. Therefore, although Lincoln had given them a motive to reak free, it was the blacks’ own courage to fight for their own freedom which ultimately led to their emancipation. The North’s victory over the South in 1865 meant that the slaves were finally free. As freedpeople, one of their foremost desires was to own land as land meant subsistence and sufficiency (Norton et al, 2008). Also, because it was all they had known to do, many fell back on plantation agriculture as their livelihood. Some, like Josua Culverson and Major X Whiteing, applied for leases through the U. S. Com of Plantations (Linden Pressly, n. ). Because most freedpeople had no money to purchase land, they could only lend it on credit. However, because of preexisting prejudices, freedpeople found even that to be a task. Consequently, they returned to their old farms where they had worked as slaves. However, unlike previously, these freedpeople sought to better their situation by reaching an agreement with the owner through a system known as sharecropping. Often, such an agreement would entail the provision of food and seeds in exchange for a portion of the crop earnings (Norton et al. 2008). Such is the case between William R. Steen, a white citizen, and Caroline, a colored woman; along with 7 other Arkansas freedpeople whom by contract, had to give one third part of the crop raised upon the farm by their labor to the owner of the farm (Linden Pressly, n. d). The years 1865 to 1877 saw efforts in reconstructing the war-torn South. A huge hurdle existed then to reconcile the freed blacks with southern whites, who were so used to operating in a slave society that their prejudices could not be eradicated so easily. Furthermore, they were threatened by the rise of black status and into the ranks of political power so much so that a group of whites came together and formed the first Ku Klux Klan; a society which sought to maintain white supremacy through intimidation, violence and terrorism (Norton et al. , 2008). Klansmen committed murder, arson, and rape whilst asserting their notion of white supremacy. As a result, despite having been emancipated, the freed blacks still suffered considerable pain and fear under the hands of the Ku Klux Klan. Amongst the most targeted were blacks who had rose to political power. Ann J. Edwards, the daughter of a black Congressman recounted that â€Å"We, his family, lived in constant fear A day or two before election a mob gathered†¦in front of the house, and we thought the end had come. † (Sterling, n. d). More gruesome was the story of Joe Johnson who was elected into the Republican office. He was burnt nearly to death, and shot because of his refusal to resign from office (Sterling, n. ). His execution was witnessed by his wife who could do nothing against the oppressive terror and violence of the Ku Klux Klan. Blacks were not the only victims of the Ku Klux Klan regime. Whites who openly supported blacks saw their lives being threatened as well. Hannah Flournoy, a black woman, gave account of an execution involving a white Georgian who had supported the black cause. â€Å"[Men] bolted right in and commenced shooting†¦They were disguised but I knew a great many of them. Hannah â€Å"thought it right to take [Ashburn] in† and was â€Å"willing to die for him† simply on the basis of him being a Republican (Sterling, n. d). The incident reflected the loyalty of many blacks to stand beside those, white or otherwise, who sought to uphold their natural rights. Teachers were another group being targeted. Colored schools came under the attack of the Klan and were burnt to the ground (Norton et al, 2008). Many blacks endured the hardship of abuse and violence despite its injustice and unlawfulness. Recounted Harriet Hernandez, â€Å"He struck me on the forehead with a pistol†¦kicked me over [the fence]†¦dragged and beat [me and my daughter] along. † Despite the blacks being freed in the South, the activities of the Ku Klux Klan ensured that blacks would not enjoy the same rights as whites. â€Å"[Colored people] have no satisfaction to live like humans†¦all summer I have been working and it is impossible for me to enjoy it†, said Harriet (Sterling, n. d). Despite all the violence inflicted on them, blacks exhibited extraordinary courage in the face of threats by Klansmen. Emeline Bremfield whose husband was a target of the Klan, stood unwavering in the face of death, as the Klan confronted her of her husband’s whereabouts (Sterling, n. d). Blacks even went out of their way to fit into a black aggrieved society. Many like, Caroline Smith and Lucy McMillan, dressed down in order not to stand out for fear of being whipped (Sterling, n. d). Although the Ku Klux Klan‘s primary motive was the assertion of white political power, Klansmen took every opportunity they got to assert white supremacy mindsets. McMillan’s house was burned simply because she had mentioned she wanted to own land. Smith was whipped only to remind her not to â€Å"sass any white ladies† (Sterling, n. d). Aside from the destruction of property and life, black women suffered in yet another form – sexual assault. Klansmen sought to assert their superiority over black women by sexually harassing them. Some black women were even mutilated, like Frances Gilmore who was â€Å"cut with a knife†, or gang raped (Sterling, n. d). Racial hostility and terror ultimately brought down the Republican regime in the South. Efforts by the Ku Klux Klan prevented strong presence of the Republican coalition in the South and a restoration of the Democratic majority. The Klan continued to terrorize black people and ran amok until the Enforcement Acts and subsequent persecutions brought an end to the first Klan. Overall, the blacks suffered greatly during the Civil War and Reconstruction Era. Despite having obtained emancipation, prejudices continued to affect their lives. Although we now know that it did not succeed in driving them out of the country, blacks today continue to face the same prejudices.

Mutant and Wild-type Yeast Strains via Mitochondria Proteins

Mutant and Wild-type Yeast Strains via Mitochondria Proteins Differentiating between mutant and wild-type yeast strains via mitochondria proteins    By: Jason Hoang Lab Partner: Daryan Chan Introduction Yeasts are important organisms due to their uses in everyday such as baking, making fermented foods and alcohol production (Steensels et al, 2014). Yeasts have been so widely studied that it was one of the first organisms to have its genome sequenced (Goffeau et al, 1996). Thus, Yeasts are more than capable of acting as a model organism for eukaryotes (Botstein et al, 2011). For this experiment we are working with Saccharomyces cerevisiae. The mitochondria is the powerhouse for cell, as it is the major production site of ATP for the cell. The inner mitochondrial space has an electrochemical gradient, from which ATP is generated by using 5 protein complexes create an electrochemical gradient to assist in ATP production (Alberts et al., 2015). The COX6 assembly accepts electrons from cytochrome c and uses oxygen as the terminal electron acceptor to make water (Alberts et al., 2015). ATP synthase then uses the resulting proton gradient made by those complexes to pump protons back into the mitochondria matrix and make ATP (Alberts et al., 2015). The objective of this experiment was to determine if a given yeast sample was a wild type or a mutant with no COX6 activity. One of the major techniques to be used in this lab is subcellular fractionation. This technique first lyses the cells and then uses centrifugal forces to separate particles by size (Alberts et al., 2015). The centrifugal forces results in the denser particles moving away from axis of rotation creating a pellet which contains the heavier particles and a supernatant which contains lighter particles (Alberts et al., 2015). Another major technique used was Gel electrophoresis. Gel electrophoresis is used so that a fraction with multiple proteins can be separated based on size and shape (Alberts et al., 2015). Protein fractions are loaded on to wells in the gel and an electrode is attached (Alberts et al., 2015). SDS page is popularly used because it can confer a negative charge and linearize proteins being run through the gel (Alberts et al., 2015). The proteins will run through the gel due to their negative charge (Alberts et al., 2015). A standard is used to provide a reference to determine the sizes of the sampl proteins (Alberts et al., 2015). One of the other major techniques used in this lab was western blotting. After proteins are run on electrophoresis a labelled antibody is exposed to the electrophoresed fractions in a process called immunoblotting in order to detect presence of a specific protein (Alberts et al., 2015). The gel is exposed to a membrane where a current is run to dive the proteins onto the membrane (Alberts et al., 2015). The membrane is then drenched in labelled antibodies to detect for a specific protein (Alberts et al., 2015). This process can detect very small amount of specific protein and is useful for detecting changes of concentration of a specific protein in a cell under various conditions (Alberts et al., 2015). To measure cytochrome oxidase activity in this lab, we looked towards Beers Law which states that the ability of a solution to absorb light at a single wavelength is proportional to the concentration of solute in solution (Lukofsky et al, 2009). This show that absorbance and concentration are linearly related. Therefore, this would allow us to determine the rate of cytochrome oxidase activity in a sample. Materials and Methods Experiment was performed according to protocols set by Department if Biology, Winter 2016, Biology 331 for Experiment 1: Subcellular fractionation of yeast cells, pg 2-8, Experiment 2: Yeast growth curve; light microscopy; protein determination, pg 1-4, Experiment 3: Polyacrylamide gel electrophoresis, pg 1-8, Experiment 4: Development of Western Blot; COX Activity Assay, pg 3-10, written by Dr. Dragana Miskovic where the experiment was performed with no deviations unless specifically noted (Miskovic, 2017) The only deviation occurred in experiment 2 where we had ran out of BSA STD and had to borrow from another group. The borrowed BSA STD was not tested to have exact concentration as specific in lab protocol and may have had different concentration. Results Experiment 1 Yeast Strain Sample: A2 Table 1. Masses recorded for Lysing Yeast Cells Section Items Mass (g) Mass of Centrifuge Bottle containing Yeast 159.2 Mass of Empty Centrifuge with Pellet 49.89 Mass of Pellet 3.23 Amount of STE solution needed to resuspend yeast pellet: 3.23g x 2= 6.46mL Table 2. Volumes recorded for the Aliquoting Yeast Subcellular Fractions Section Solutions Total Volume LPS 3mL + 3.7mL= 6.7mL HSS 3mL + 2.8mL= 5.8mL MITO 300ÃŽÂ ¼L + 300ÃŽÂ ¼L= 600ÃŽÂ ¼L It was also noted that after the MITO fraction was made the pellet was intact and not messy Figure 1: Drawings of 50/100/200 ÃŽÂ ¼L dye drops from pipetman and expected 1 mL dye drop Experiment 2 Part A Table 3. Concentration of Yeast Cell at Two Different Times Time OD600 Reading Concentration (cells/ mL) 2:47pm 0.021A 210,000 4:59pm 0.043 A 430,000 It has been determined that an OD600 value of 1.0 is thought to contain roughly 1 x 107 cells/ mL. An OD600 value of 0.021 A will contain a concentration of 210 000 cells/ mL. An OD600 value of 0.043 A will contain a concentration of 430 000 cells/ mL. Figure 2. This graph shows the change in absorbance of yeast culture at 0 and 120 minutes. Equation to represent growth is calculated and shown above. Calculating Doubling time Formula for growth of yeast is y=0.0002x + 0.021. given initial absorbance reading of 0.021, doubled concentration should give reading of 0.042. Therefore use y= 0.042, where x means time in minutes 0.042=0.0002x + 0.021 where x = 105. Therefore it was found that doubling time is 105 minutes. Part B Yeast cells dyed with methylene blue stain Figure 3. These are some of the cell types that were observed when the overnight culture was stained with methylene blue under 40x magnification. It was found that roughly a third had a stained positive for a nucleus. None of the cells appear to be multi-nucleate. No vacuoles were observe either Yeast cells dyed with neutral red Figure 4. These are some of the cell types that were observed when the overnight culture was stained with neutral red under 40x magnification It was found that over 90% of the cells stained positive for a nucleus. Many of the cells appeared to be multi-nucleate and budding as well. It appeared that 1 or 2 vacuoles appeared to be detected per cell. Part C Table 3. Absorbance Results for the BioRad Protein Determination Assay 1 2 3 4 5 6 7 8 9 10 A 0.27 0.191 0.21 0.201 0.196 0.37 0.397 0.404 0.369 0.036 B 0.246 0.24 0.303 0.192 0.226 0.245 0.272 0.372 0.252 0.035 C 0.23 0.263 0.248 0.294 0.037 0.036 0.036 0.036 0.036 0.036 D 0.256 0.227 0.25 0.277 0.035 0.035 0.035 0.035 0.035 0.034 E 0.246 0.182 0.242 0.215 0.474 0.362 0.306 0.389 0.482 0.035 F 0.289 0.349 0.285 0.246 0.299 0.264 0.347 0.738 0.203 0.036 G 0.203 0.254 0.321 0.249 0.035 0.035 0.035 0.037 0.035 0.036 H 0.2 0.261 0.263 0.274 0.034 0.034 0.035 0.035 0.035 0.034 11 12 A 0.036 0.037 B 0.035 0.035 C 0.037 0.038 D 0.035 0.044 E 0.036 0.035 F 0.035 0.035 G 0.035 0.035 H 0.035 0.034 Figure 4. This graph shows absorbance readings of standard solution using BSA at different concentrations. Calculating concentrations of LSP, HSS, and Mito. Equation for concentration of solution based on absorbance reading was determined based on above graph. Equation yielded was y=0.1264x + 0.2159 Average absorbance readings: LSP was 0.245, HSS was 0.249, and MITO 0.289. Using found readings as y for above equation we calculated concentration of proteins in each sample. LSP: 0.245 = 0.1264x + 0.2159Therefore x = 0.230 mg/mL HSS: 0.249 = 0.1264x + 0.2159Therefore x = 0.262 mg/mL MITO: 0.289 = 0.1264x + 0.2159Therefore x = 0.551 mg/mL Dilution factor needed to get fraction to 2ÃŽÂ ¼g/mL LSP: (0.230 mg/mL)(0.1mL)=> (0.0230 mL)(1/x mL)(10 dilution factor) = 2mg/mLTherefore x= 0.115 mL HSS: (0.262 mg/mL)(0.1mL)=> (0.0262ÃŽÂ ¼g)(1/x mL) (10 dilution factor) = 2mg/ mL Therefore x= 0.131 mL MITO: (0.551 mg/mL)(0.1mL)=> (0.0551ÃŽÂ ¼g)(1/x mL) (10 dilution factor) = 2mg/ mLTherefore x = 0.2755 mL Experiment 3 Figure 5. PVDF Membrane after proteins are transferred over from gel after electrophoresis. Our group is left side (D.C, J.H) Experiment 4 Figure 6. Membrane after detecting solution had been added over 10 minutes ago. Bands on right hand side are the standard Figure 7. This graph shows the distance travelled by each protein in the standard mix against the Log(Mw) on semi log paper Table 4. Cytochrome c Oxidase (COX) Activity Assay Sample Absorbance At 0 sec (OD) Absorbance after 20 sec (OD) Change in absorbance Change in Concentration (ÃŽÂ ¼mol/mL) COX activity (ÃŽÂ ¼mol/ L/min) Blank 0.525 0.525 0 0 0 LSP (1) 1.259 1.253 0.006 0.2143 0.6429 LSP (2) 1.272 1.264 0.008 0.2857 0.8571 HSS (1) 0.493 0.491 0.002 0.0714 0.2143 HSS (2) 0.496 0.491 0.005 0.1786 0.5257 MITO (1) 0.553 0.557 -0.004 -0.1429 -0.4286 MITO (2) 0.537 0.535 0.002 0.0714 0.2143 Table 4. This shows the Sample calculation for COX activity Change in absorbance = Absorbance at 0 sec Absorbance after 20 sec 1.259-1.253 = 0.006 Change in concentration ΆA = ÃŽÂ µ x b x Άc 0.006 = 28mM-1-cm-1 x 1 cm x ΆcTherefore Άc = 0.0002143 mM => 0.2143 ÃŽÂ ¼molAssuming volume of assay is 1.0 mL, change in concentration is 0.2143 ÃŽÂ ¼mol/mL COX activity COX activity = change in concentration / time 0.2143 ÃŽÂ ¼mol/mL / (1/3 min) = 0.6429 ÃŽÂ ¼mol/mL/min Figure 8. Graphical representation of COX activity in LSP fractions Figure 9. Graphical representation of COX activity in HSS fractions Figure 10. Graphical representation of COX activity in MITO fractions Discussion Galactose was used over glucose as a carbon source for our yeast cells. This is because we wanted to determine if the mitochondria was functional in our yeast cells. Different yeast strains will use different metabolic pathways in presences of each. When glucose is used as a carbon source the yeast cells will generate ATP via fermentation, whereas when Galactose is used the cell will perform oxidation. This is important to observe as different yeast strains will have varying levels of cytochrome c usage based on that. To visually determine if cytochrome c will be utilized by the cell we can look at the fractionation experiment earlier. When separating for the MITO fraction if one had found a messy pellet it would have indicated that the mitochondria was not intact while solid pellets would indicate the mitochondria was intact. If one did find a messy pellet it could have been the result of differences in fractionation techniques, cells being lysed prior, or something had disturbed the cell in transport. For our experiment we had found the mitochondria to be intact, which is a strong indicator that the mitochondria for our sample was present. To reach cytochrome c oxidase (COX) we used diferential centrifugation which seperates objects based on size and density, where larger molecules such as the intact cells will settle at the bottom of a tube while mitochondria which is smaller would remain in supernatant. This is also why we had separate centrifugations, to get samples with intact cells and samples with intact mitochondria. Density gradient centrifugation is also a widley used technique that seperates based on density. In that case we would see multiple bands form in tubes with densest molecules gathering at the bottom and bands above it with less dense molecules. Experimentally we found yeast doubling time to be 105 minutes (1.75 hours) when inoculated in YPD (1% yeast extract, 1% peptone, 2% glucose). It has been determined in many other experiments that Saccharomyces cerivisiae has a doubling time of 1.69 hours (Deak, 2008). The difference could be attributed to many factors such as environment (amount of light, heat, and etc) and growth substrates used. But the difference is not very large and would still be considered to conform to literature results. During the methylene blue staining of yeast cells it was noted that roughly a third of yeast cells contained a nucleus but it did not seem to be multi nucleate. While the neutral red stains showed that many cells appeared to be budding with one or 2 vacuoles present per yeast cell. These findings fall in line with what is normally expected from yeast cells as they do have vacuoles in their cells (Armstrong, 2010). Furthermore results also fall in line with yeasts having nucleuses but not being multi nucleated (Roberts and Ganesan 1959) One thing that may have affected a major portion of the experiment was determining the concentration of each respective LSP, HSS, and MITO fraction and diluting it to 2mg/mL. it is important to note that during pipetting steps to get each sample that the suspensions be homogenous beforehand otherwise you may be taking up different components of the fraction and missing others depending on how deep the pipet was inserted. During the remainder of the experiment it was found that after gel transfer to PVDF membrane and during western blotting that very few to no proteins were showing up. If low concentration of protein was a factor then it would most likely be traced back to this step. Many reasons can be attributed to this for instance, poor pipetting technique, the fractions were not homogenized properly before pipetting or even the dilution factor could have been incorrect. As noted during the material and methods during the preparation of experimental samples which would to create o ur protein concentration standard curve we had run out of BSA STD and required taking some from another group. When we created our protein concentration standard curve it came out completely odd, having unexpected drops in absorbance readings. The expected result was a linear curve where a higher BSA STD concentration would have led to a higher absorbance readings. Due to the change in BSA STD this may have had a different concentration due to being taken from a different location in its container it could have had a different concentration. Thus causing inconsistencies for our standard curve. As the standard curve was deemed incorrect afterwards any protein concentration calculations based on it would have been flawed, leading to incorrect dilutions. If the dilutions been calculated incorrectly, as they most likely were, there is the chance that the protein fractions would have been over diluted leading to not enough protein to be present for visible bands for the gel electrophores is and western blotting. For the gel electrophoresis SDS was included in solubilisation buffer to give proteins inserted into the wells a negative charge so that when a charge was applied they would run to the other end of the gel and to help unfold the protein so that it would be able to go through the gel. For this experiment a 12% gel was used in the interest of saving time because a 15% gel would have caused the proteins to go through it slower leading to a lower resolution of identifiable protein bands. The purpose of transferring proteins from gel to PVDF membrane was to be able to visualize the movement of proteins on the gel after electrophoresis. To accomplish this we applied Ponceau stain to the membrane to increase the resolution of the bands and to ensure equal amounts of proteins are loaded onto the gel (Al-Amoudi et al., 2013). It was found that after proteins had been transferred to PVDF membrane that we had very few bands show up for the solubilizing buffer lane, both LSP sample lanes, and both HSS sample lanes. Bands did appear for both MITO samples, however, it appears that got smeared across the gel, bleeding over to other wells. This could have been the result of diluting samples in the wells for reasons noted above, the SDS gel would have been poorly constructed and contributed to the smearing, and poor electrode contact on the gel might have blocked the gel from having proper electrical charge. Issues could have also arisen during transfer of proteins from gel to me mbrane. Air bubbles could have been present during transfer which would have prevented any protein from being transferred as proteins cannot move through air. Additionally, poor folding between membrane and the gel could have attributed to smearing of MITO samples. The purpose of the western blotting was to be able specifically detect for the presence of biotinylated COX proteins. In order for a cell to express a biotinylated protein it needs to be able to take up foreign DNA, be able to properly fold COX-biotin fusion protein, the cell needs to be able to recognize the BSS signal fused to C terminus, and be able to translate COX and biotin together (moving stop codon so that it doesnt not stop halfway across the other.) It was found that after western blotting our membrane with protein fractions that no bands had appeared even after 10 minutes of membrane being in contact with detecting solution. This led to Figure 7. The chart showcasing the relative distances that proteins have travelled is blank as a result. This would imply that when the blocking solution was added that it managed to block the entire membrane (and any present proteins included) from interacting with the probe. However Tween-20 was used to wash excess reagent. So the milk most likely would not have been able to bind to any protein after introduction of Tween-20. Therefore the lack of data could be attributed to low concentrations of protein on membrane for reasons as noted above. Referring to Figure 5 the only proteins that were found on the membrane after were MITO which shows that there would have been no LSP or HSS for probe to bind to, whereas for present MITO sample the concentration may not have been high enough and as a re sult some of it could have been washed out by the methanol step causing concentration of MITO to be so low that it could have been blocked by the blocking solution. There is also a possibility that our yeast samples were not able to biotinylate the COX protein at all which could explain why there were no bands occurring Looking at COX activity graphs for LSP, HSS, and MITO they seem to follow what is expected except for HSS. COX was used as an identifying marker for identifying subcellular fractions containing COX because it is an integral membrane protein for the inner membrane space. If COX activity is present then that would indicate that the mitochondria is intact and functioning. These samples should have seen increased COX activity as cytochrome c was introduced into the fractions which provide electrons to the COX protein allowing it to pump proteins and reduce oxygen to water. Both MITO and LSP experienced increased COX activity as shown by figure 8 and 10 respectively. This falls in line with what was expected with the MITO fractions experiencing higher levels of COX activity then the rest as the cytochrome c had less of a distance to travel to reach inner mitochondrial membrane space than LSP. LSP should have a signal because it would contain intact yeast cells which have mitochondria Albe rts et al., 2015). Therefore LSPs rate of COX activity should be lower because the cytochrome c would have harder time reaching mitochondria. This is shown by Figure 10 having steeper reaction times than Figure 8 and 9. This reaction utilized Deoxycholate (DOC) to speed up the reaction which is why it was only done in 20 second intervals as DOC solubilizes with cytochrome c so that it can enter the mitochondria to interact with COX. If reactions were tested too long after DOC was added then the reaction would have finished before being able to measure absorbance. The one that did stand out was the HSS fraction which appeared to experience negative COX activity or none at all. This was expected as it should have all the remaining parts of the cell that werent the mitochondria, lysosomes and peroxisomes Alberts et al., 2015). This would indicate that these samples did not have an intact mitochondria with a COX protein to interact with cytochrome c. this could be explained by the In conclusion it was found that our yeast strain A2 is the wild type strain. This is because during initial centrifugation the resulting pellet was solid indicating intact mitochondria. Furthermore during COX assay the MITO strain indicated an active COX as shown by its increase in activity, proving that A2 was in fact a wild type strain with functioning mitochondria. References Al-Amoudi, M.S., Salman, M., Al-Majthoub, M.M., Adam,Abdel Majid A., Alshanbari, Naif A., Refat, Moamen S., (2013) Res Chem Intermed 41: 3089. doi:10.1007/s11164-013-1417-4 Alberts, B. et al. (2015). Molecular Biology of the Cell Sixth Edition. New York, NY: Garland  Science, Taylor Francis Group. Armstrong, John. Yeast vacuoles: more than a model lysosome. Trends in Cell Biology 20.10 (2010): 580-85. Web. 13 Mar. 2017. Botstein, D., Chervitz, S. A., Cherry, J. M. (1997, August 29). Yeast as a Model Organism. Retrieved March 12, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039837/ Deak, Tibor. Handbook of Food Spoilage Yeasts, Second Edition. Contemporary Food Science (2007): 50-51. Web. 13 Mar. 2017. Glerum, Moira Miskovic, Dragana (2017). Biology 331 Advanced Cell Biology Laboratory Manual Winter 2016. Biology Department, University of Waterloo, Waterloo. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD,  Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG  (1996) Life with 6000 genes. Science 274, 546 Lukofsky, David, Jonathan Bessette, Heejeong Jeong, Elsa Garmire, and Ulf ÃÆ'-sterberg. Can precursors improve the transmission of energy at optical frequencies? Journal of Modern Optics 56.9 (2009): 1083-090. Web. 13 Mar. 2017. Roberts, C., and A. T. Ganesan. The occurrence of multinucleate giant cells in yeasts. Antonie van Leeuwenhoek 25.1 (1959): 97-107. Web. 13 Mar. 2017. Steensels, Jan, Tim Snoek, Esther Meersman, Martina Picca Nicolino, Karin Voordeckers, and Kevin J. Verstrepen. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiology Reviews 38.5 (2014): 947-95. Web. 13 Mar. 2017.

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