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Units Of Galactosidase Enzyme Biology Essay

Units Of Galactosidase Enzyme Biology Essay. Amongst the bacteria, the bacterium E. coli has an efficient mechanism for metabolising lactose. Three proteins that are important in lactose metabolism are all encoded in a single expressible unit of DNA, called the lac operon. The lac operon consists of three structural genes, a promoter, a terminator, regulator and an operator. The structural genes in lac operon encode three types of proteins which are involved in the metabolism of lactose in the E. coli. They are: β -galactosidase; involved in converting lactose into glucose and galactose, β -galactoside permease; responsible for transporting lactose into the cell and finally β -galactoside transacetylase which its function is not yet known. The bacterium does not waste energy expressing these proteins if lactose is not present in the growth medium. It only makes these proteins when lactose is available to be metabolized. In an E. coli cell growing in the absence of lactose, a repressor binds to the operator, preventing RNA polymerase II from transcribing the lac operon’s genes. The operon is OFF. When an E. coli cell is growing in the presence of lactose the inducer, allolactose; which is the product of lactose binds to the repressor and prevents it from binding to the operator region. As long as there is no repressor to bind to the operator, RNA polymerase can recognize the promoter and start transcribing the structural genes in the lac operon. So, this experiment is aiming to investigate the expression of lac operon under different condition such as presence of an inducer such as IPTG, alternative source of energy to lactose such as glucose as well as presence of bactericidal antibiotics which prevents protein synthesis in E. coli. The following experiment was carried out to examine the relative levels of B-galactosidase in growing E. coli K12 cells using B-isopropylthiogalactoside (IPTG) as the inducer and compares this to the situation with no induction as well as determining the induction time of the lac operon in E. coli . Moreover, this experiment will investigate the effects of glucose, chloramphenichol, rifampicin and streptomycin addition to the system as together with a comparison of lactose as the inducer rather than IPTG. Material and methods: In order to carry out this experiment, the materials and experimental procedures described in BIOC2201 manual were followed carefully without introducing any changes to it. These include: The experimental procedure for investigating the course of induction of β-galactosidase by IPTG experimental procedure, p 50-51. Experimental methods for the assay of β-galactosidase activity, p 52-55. The experimental procedure for investigating the characteristic of the induction of β-galactosidase, p 56-58. Result: Table A: AVERAGE ABSORBANCE READING BEFORE AND AFTER SUBTRACTION OF BLANK (IPTG, CONTROL). GRAPH A (The graph of Units β-galactosidase per ml of bacterial culture against the time of induction with IPTG) Worked example of calculation: The number of Units of β-galactosidase enzyme in each tube was calculated as following given that one unit of β-galactosidase is the amount of enzyme that catalyse hydrolysis of 1micro mole of ONPG to o-nitrophenol per minute, and the ϵ414 of o-NP is 21,300 M-1cm-1, the path length is 0.9 cm and the assay volume is 0.8ml. The number of Units of β-galactosidase enzyme in tube 1 (induction time 1). Assay volume = 0.8ml = 0.0008L A = ϵCl C = A/ϵl C = (0.003)/(21,300M-1cm-1 x 0.9 cm) = 1.56 x 10-6 = 1.56 µM Now using n=cv, the number of moles of o-NP in 0.0008L of assay is: N = cv N = 1.56µM x 0.0008L N = 1.25 x 10-2µmol As the induction time was five minutes, the number of Units of β-galactosidase can be calculated as: Units of β-galactosidase = 1.25 x 10-3µmol/5min = 2.50 x 10-4µmol/min As this is the Units of β-galactosidase in 200µL of bacterial culture, now, the Units of β-galactosidase per one ml of bacterial culture would be five times greater, i.e. 2.50 x 10-4µmol/min x 5 = 1.25 x 10-3µmol/min The same method was used to calculate the number of Units of β-galactosidase in the other tubes. TABLE B: AVERAGE ABSORBANCE (A414) READING BEFORE AND AFTER SUBTRACTION OF BLANK (IPTG, IPTG CHLORAMPHENICHOL) “sourced from my own experiment results” This result was not used for TABLE C: AVERAGE ABSORBANCE (A414) READING AFTER SUBTRACTION OF BLANK (sourced from the Blackboard model results) Worked example of calculation: The number of Units of β-galactosidase enzyme in each tube was calculated as following given that one unit of β-galactosidase is the amount of enzyme that catalyse hydrolysis of 1micro mole of ONPG to o-nitrophenol per minute, and the ϵ414 of o-NP is 21,300 M-1cm-1, the path length is 0.9 cm and the assay volume is 0.8ml. The number of Units of β-galactosidase enzyme in tube induced only by IPTG and labelled as 5 (induction time 5) can be found as following: V = 0.8ml = 0.0008L A = 0.088 A = ϵCl C = A/ϵl C = (0.088)/(21,300M-1cm-1 x 0.9 cm) = 4.59 x 10-6 M = 4.59 µM Now using n=cv, the number of moles of o-NP in 0.0008Lof assay is: N = cv = 4.59µM x 0.0008L = 3.67 x 10-3µmol As the induction time was five minutes, the number of Units of β-galactosidase can be calculated as: Units of β-galactosidase = 3.67 x 10-3µmol/5min = 7.34 x 10-4µmol/min As this is the Units of β-galactosidase in 200µL of bacterial culture, now, the Units of β-galactosidase per one ml of bacterial culture would be five times greater, i.e. 7.34 x 10-4µmol/min x 5 = 3.67 x 10-3µmol/min The same method was used to calculate the number of Units of β-galactosidase in the other tubes. TABLE D: Units β-galactosidase per ml of bacterial culture operon in the presence of IPTG, IPTG Chloramphenichol, Lactose, IPTG “5 minutes” glucose, IPTG “10 minutes” glucose, IPTG rifampicin or IPTG streptomycin) GRAPH B (The graph of Units β-galactosidase per ml of bacterial culture against the time of induction of lac operon in the presence of IPTG, IPTG Chloramphenichol, Lactose, IPTG “5 minutes” glucose, IPTG “10 minutes” glucose, IPTG rifampicin or IPTG streptomycin) Discussion: The amount of β-galactosidase production in each tube is the indictor of expression of lac operon in that tube. Hence, quantifying the amount of β-galactosidase in tubes, each under different condition is a very useful tool to understand the role of inducers and repressors in controlling the lac operon expression and in general, the control of gene expression in prokaryotes. At the given time sets, CTAB was added to the tubes to kill the E. coli cells and lyse the cells to release its contents including galactosidase enzyme. As it is difficult to directly quantify the amount of β-galactosidase produced in the tubes, but we can measure id indirectly. Î’-galactosidase can convert ONPG to galactose and o-nitrophenol which has a yellow colour with an absorbance maximum at 414nm. The absorbance reading in each tube indicates the amount of ONPG converted to galactose and nitrophenol by the enzyme β-galactosidase. This in turn indicates the amount of β-galactosidase enzyme produced in each tube, i.e. the greater the absorbance reading, the greater the amount of β-galactosidase in each tube. Using this method, the rate of production of β-galactosidase, hence the rate of expression of lac-operon in E. coli in the presence of inducer, effectors and repressors were investigated. Graph A shows the amount of β-galactosidase produced in E. coli after adding the IPTG; the inducer in different period of times. At first glance, it can be seen that the graph does not cut the X axis at time 0 when the IPTG and CTAB were added at the same time. This is because the IPTG did not have enough time to induce the lac operon. The graph cuts the X axis at approximately 2-minutes after the addition of inducer (IPTG). This is when there was significant amount of β-galactosidase produced, i.e. the lac operon was first induced. This suggests that adding an inducer to the E. coli cell does not lead to an immediate production of β-galactosidase; rather it takes a while for the lac operon to pass all the expression stages and produce the final product; β-galactosidase and other enzymes. Looking at the control, there seems to be no β-galactosidase made in the process. This is because there was no IPTG added to the tubes and the repressor was not inhibited from binding to the operator, hence the RNA Polymerase II did not bind to the operator to initiate the transcription process. Looking at the graph B, (IPTG only) there is a positive linear relationship between the time of induction with IPTG and the amount of β-galactosidase production in the tubes. IPTG is an inducer, binding to the repressor protein and inhibit its binding to the operator region in the lac Operon and allows the lac-Operon itself to be expressed. Moreover, in the tubes where the lactose was added, the amount of production of β-galactosidase was quite low compared to that of where there was no lactose present. When there was lactose added to the tube, it was converted to products; allolactose and glucose by the enzyme β-galactosidase. Allolactose which has a similar action to IPTG, act as inducer, binding to the repressor and inhibits it’s binding to the operator region in the lac operon and allows it to be expressed. Allolactose unlike IPTG can be easily hydrolysed so its inducing action lasts for a shorter period of time compared to that of IPTG. As a result, its graph looks flatter. When the lac operon expression was investigated in the presence of glucose, the graph initially increased but once the glucose was added, the graph started to flatten out. Lactose is not the preferred source of energy in E. Coli cell. If lactose and glucose are present, the cell will use all of the glucose before the lac operon is expressed. To prevent lactose metabolism, a second level of control of gene expression exists. The promoter of the lac operon has two binding sites, one for RNA polymerase enzyme binding and the other site is where catabolite activator protein (CAP) and cyclic AMP (cAMP) bind to. For the lac operon to be transcribed, it is crucial for the CAP-cAMP complex to bind to the promoter site. On the other hand, wether this complex is present in the cell or not, is associated with the availability of glucose inside the bacterial cell. When there is high amount of glucose present n the cell, the amount of cAMP decreases and vice versa. Once the level of cAMP was declined, this lead to the inactivation of CAP RNA Polymerase II complex. This inactivation in tern inactivates the promotor, hence the lac operon is getting turned off. Hence, after the time 5 minutes, the lac operon was repressed. In the tube where the glucose was added at the time 10 minutes, the graph started to flatten out right after adding the glucose. Here, the E. coli had more time to express the lac operon and make more of β-galactosidase enzyme. These two stages of the experiment show that glucose is of a major important in controlling the lac operon expression in E. Coli. On the other hand, looking at the graph B, where there was chloramphenichol added to the tube at the time 10mins, the rate of production of β-galactosidase remained almost constant after the time of addition. This is because chloramphenichol is an antibiotic, inhibiting the peptidal transferase activity of the bacterial ribosome, hence blocking elongation of polypeptide chain, i.e. β-galactosidase enzyme. Similarly, rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells by binding its beta-subunit, thus preventing DNA transcription and hence protein syntheses. Streptomycin, another antibiotic is also protein synthesis inhibitor. It binds to the 30S subunit of the bacterial ribosome, interfering with the binding of tRNA to the 30S subunit. This action blocks translation of lac operon mRNA in E. coli and prevents the β-galactosidase enzyme to be made. These types of actions of antibiotics block the expression of lac operon and keep the amount of β-galactosidase enzyme to the level which was present in the tubes before the antibiotics addition. This is clearly shown in the graph B. This inhibition of lac operon by antibiotics indicates that the level of production of β-galactosidase is regulated by the expression of lac operon. Conclusion: Finally, from this experiment it can be concluded that the expression of Lac operon in E. coli is being controlled regularly depending on the availability of lactose in the cell, presence of inducers and the availability of alternative source of energy for metabolism instead of lactose. When there is glucose present in the cell, the lac operon is being repressed. Likewise, in the presence of lactose and the absence of glucose, the lac operon is being expressed to metabolise lactose molecules in the cell. Overall, these finding suggests that the bacterial cells are very efficient in terms of energy consumption; avoiding spending energy to produce unnecessary proteins. Units Of Galactosidase Enzyme Biology Essay

Designing a Structure

Designing a Structure. I’m working on a Management question and need guidance to help me study.

Key questions to ask when designing an effective organizational structure include:

How many individuals can a manager direct efficiently and effectively?
Where should decision-making authority lie – entirely with the manager or more as collaboration between manager and staff?

Answer each question in 2-3 paragraphs based on personal experience
and your readings. Be sure to use proper spelling, punctuation, and
grammar and cite your sources per APA.
Designing a Structure

Political Views on Slavery in the US

custom essay Mark Dawod Political Compromise DBQ Economics, politics, and society played the biggest roles when it came to making the compromise of slavery agonizingly difficult for the north and south. Economics played its role when it came to making compromise between the two opposing forces difficult. For one, the South’s society depended on slavery to make their economy prosper, it was basically the foundation to their entire economy. So much so that they resented a free society (Doc 6). Herald, who was quoted in the New York Tribune in 1856 stated, “Free society! We sicken at the name,” in which he would go on bashing the north along with their “greasy mechanics and filthy operatives.” The purpose of this article was to show the South’s hatred toward a society without slaves, which is reliable because without those men laboring in their fields, they would all be living in poverty. Although the North’s economy was prospering and catching up, it was nothing compared to the South’s economy, ranked number four worldwide. During the Second Great Awakening, antislavery movements became more and more common, which angered the South. The Declaration of the National Anti-Slavery Convention (Doc 2) wanted to abolish slavery, saying that all laws allowing slavery would therefore be null in void before God. Their purpose for writing this report being that slavery was morally wrong, and therefore it should be terminated once and for all, also since this was written by a small group of people for all to read, I think it was fairly accurate concerning their true beliefs on slavery. This was something the South surely would not have agreed with, however, the Resolution of the Pinckney Committee (Doc 3) was more likely approved by them as it was more like their mindset. Pinckney’s Committee’s Resolution was to keep any further action against slavery from taking place, basically they did not want any more petitions, memorials, propositions, etc. relating to slavery. This document’s audience, being the house of representatives, and Pinckney’s background with South Carolina, makes this document biased because he was likely a slave owner himself. Differing political views also made compromise difficult. For example, the imbalance of states would lead to angry southerners, or northerners, and cause much more devastating occurrences, for instance, Bleeding Kansas. Popular Sovereignty was strongly supported by the South, and when it was declared that Kansas would not be a slave state, Southerners resented and went ahead making their own legislature in the state, which eventually led to the death of many persons who inhabited Kansas. This wasn’t the only time the South resented the federal government. Senator Henry Clay of South Carolina, in his speech to the Senate (Doc 1), argues that South Carolina has the right to defeat certain laws it deems unconstitutional. Since South Carolina believed so heavily in states’ rights, and that their audience was the Senate, it was likely very biased to support their motives in keeping their slaves and not being absurdly taxed. In Daniel Webster’s speech to the Senate (Doc 4), he attempts to speak as an American, he sides with the south when it came to the North not fully fulfilling their constitutional duties because they refused to follow the Fugitive Slave Act. He also went on to describe how it would be morally impossible to separate the north and south. Since Daniel Webster took the side of both forces, and had the point of view of an American citizen, this source can be considered reliable in viewing the situation from both halves of the country. In Abraham Lincoln’s speech at Alton, Illinois (Doc 7) he attempts to defend politicians describing how northern politicians and officiers shouldn’t be blamed for this difficulty regarding the issue of slavery, but this same power that operates in the minds of these men, is also all around them, in books, religions, and morals. One of these books being Uncle Tom’s Cabin. Society and the differing beliefs among the common people also played a big role in making compromise difficult, Uncle Tom’s Cabin, a true story about a slave’s experience in the South sparked a major outbreak in antislavery believers, their motives to end slavery suddenly became stronger. The Dred Scott decision also had differing views, for example, the Northern abolitionists saw this as a conspiracy, being that the South had set this up to forever keep slavery in their society as these African American men had no constitutional rights as they were not even citizens, not only that, but they were considered white man’s property. The South, however, applauded this Supreme Court decision, as once and for all-or so they thought-they would be able to keep their slaves. Depicted in the illustration in Document 5, Sumner is being attacked by Brooks for verbally attacking Democrats, who the south despised. The purpose of this illustration was likely to depict how the South would result to such childish actions to get what they desired. Brooks wasn’t just applauded by the South, he was praised, which would make this illustration a reliable forefront to the South’s internal motives. This difficulty in compromising between two differing forces can also relate back to Britain’s control over the colonists. They wanted, by any means, to tax the colonists as they thought they had the right to do whatever they wanted since the colonies basically belonged to them. This connects back to the issue of slavery and coming to a compromise because each side tried to make it so that they had control and keep what they desired, they would even come to such desperate actions such as starting battles and hitting one another with canes, just as the colonists kept moving west of the proclamation line and the british soldiers being given the right live in any colonist’s house. Both situations would also eventually lead to a war, required to make peace.

3 Pages Essay MLA FORMAT

3 Pages Essay MLA FORMAT.

Purpose and Overview
The second out-of-class assignment of this semester adds to the skills you have already been developing by requiring the use of information in your readings for a specific purpose. In College Writing (ENGL 100 F), you are often asked to address ideas you have found in readings. You will need to be selective in your choice of an idea from the readings to analyze and/or explain.
Background Readings (in American Mashup)
Jeffrey Zaslow, “The Most Praised Generation Goes to Work,” pp. 550-556
Jamie Pritscher, “Yes, I’m 26. And Yes, I Do the Hiring,” pp. 557-560
Daniel Brook, “Usury Country: Welcome to the Birthplace of Payday Lending,” pp. 568-575
Barbara Ehrenreich, “Nickel-and-Dimed: On (Not) Getting By in America,” pp. 577-591
John Richardson, “The Tao of Plumbing: No Pipes, No Civilization,” pp. 593-600
Writing Task 
Choose a key point made in one of the assigned readings to discuss in an essay of at least three pages. Answer the following questions as your primary focus:
How accurate is the key point made by the writer? Why is this point accurate or inaccurate, valid or not? 

You must limit your discussion to one specific point made in the assigned reading that you choose to discuss, not all of the points that the writer makes. Keep the emphasis on a main idea rather than on a minor point or detail. 
Early in your essay, you will need to summarize the overall reading and, in particular, the key point that it makes that you’re going to discuss. This provides a context for discussion of the idea you choose but limits the amount of attention given to the rest of the reading. 
You may use additional outside sources to help you make your points, but you are not required to do so for this essay. That is optional. 

Essay 2 Rubric.docx
3 Pages Essay MLA FORMAT

Analysis of experience: Clifford and Chance Essay

Table of Contents Introduction Motivation and organisational culture Components of organizational culture Employee motivation and its relation to culture Conclusion Reference List Introduction Organisations consist of different individuals that deal with disparate aspects that institutions require in order to increase profitability and ensure the smooth running of operations. In order to ensure there is order in the performance of every day tasks, most institutions set up an administrative hierarchy. The essence of the hierarchy is to facilitate a concise flow of commands from the top management to the lowest ranking employees and accountability and responsibility by every individual. However, certain issues arise regarding the execution of commands and the effects they have on the organisation. For instance, leaders have to ensure the fulfilment of a company’s goals through their actions at the work place and their formulae for decision-making and governance. Employees play their part in the productivity of a company by ensuring that their work is of high quality and that it is done in time and in accordance with the leaders’ instructions. This essay looks at issues regarding organisational culture, motivation, and teamwork coupled with the importance that each of these aspects bears in an organisation in relation to success and fulfilment of company goals. It explains some of the theories that contribute to these subjects and looks at the practical implications of these theories in relation to the every day operations of a company using Clifford Chance, a law firm based in London, as a case study. Motivation and organisational culture According to Friedman (2002), it is the moral obligation of every business to make profits. Various individuals working at a company in their different capacities thus have to ensure that their roles contribute to the overall productivity of the company. The task of ensuring that the company’s productivity remains high without compromising on the welfare of the employees and the organisation’s culture lies with the manager and at times this task may prove to be complex. One of the difficulties that the situation creates is the formulation of a form of balance between the three elements (Montana

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