Induction of Beta Galactosidase in E.Coli | Lab Report

Induction of Beta Galactosidase in E.Coli | Lab Report Aim: To study the effects of IPTG, lactose, glucose, chloramphenicol, rifampicin and streptomycin induction on the units of beta-galactosidase of E.coli Introduction In 1961, a special group of units that able to control its beginning and ending of transcription activities by undergoing induction process was discovered by Jacob and Monod. This special enzyme is known as Lac operon which is group of genes that arranged in sequences of promoter region, operated region and structural genes that found in the Escherichia Coli. Promoter region is the site where the RNA polymerases binds to and initiate the transcription process while the operated region in the operon is the site where the regulatory protein such as inducer and repressor bind to and stimulate the genes to turning on or off the transcription process. Lac operon made up of three specific lac genes; there are lac Z, lac Y and lac A and they can be found in the structural genes of the operon. At the end of the transcription process, Lac Z gene can be encoded into ÃŽÂ ²-galactosidase which is an enzyme that used to hydrolyze lactose molecules into allolactose molecules fist then further into glucose and galactose which are the monosaccharide molecules (Kathryn Grace Patterson, 2009). Allolactose is the intermediate product when ÃŽÂ ²-galactosidase used to catalyses the reaction of converting lactose to glucose and galactose. According to Miiller-Hill, Rickenberg Wallenfels, allolactose is a natural and effective inducer to trigger transcription process to occur (1964). lacY encodes into ÃŽÂ ²-galactoside permease which playing the role in transporting lactose molecules into the cell while lacA is converted into ÃŽÂ ²-galactoside transacetylase through transcription process which is an enzyme that involved in adding an ac etyl group (CH3) from acetyl coenzyme A to the 6 position of the ÃŽÂ ²-galactosidase (Xing Guo, Wang, Laurence R. OlsenSteven L. Roderick, 2002). As the inducer such as allolactose (natural inducer) or IPTG, Isopropyl ÃŽÂ ²-D-1-thiogalactopyranoside which is an artificial inducer binds to the operated region and deactivated the repressor protein. Hence, repressor protein generated by the lac I gene cannot binds to the operated region which stimulates the RNA polymerases to bind to the promoter region and start the transcription process which is positive control mechanism. On the other hand, negative control mechanism occurs when active repressor protein binds to the o-site (operated region), it blocks the RNA polymerases binds to the p-site (promoter region) and thus no transcription can take place. IPTG acts as the inducer due to its structure is similar to the allolactose. o-nitrophenol ÃŽÂ ²-galactosidase Isopropylthiogalactosidase (IPTG) ortho-Nitrophenyl-ÃŽÂ ²-galactosidase  which known as ONPG is used to measure the ÃŽÂ ²-galactosidase enzymatic activities in this experiment which show a yellow colour when ÃŽÂ ²-galactosidase  presence. ONPG has a similar structure as lactose which also catalyze by the beta-galactosidase enzyme to form galactose + O-nitrophenol whereas the O-niotrophenol responsible to the colour changes. When the intensity of yellow colour increases, the rate of enzymatic activity also increases. Hans Noll and Joseph Orlando also mentioned that o-nitrophenol-beta-galactosidase is hydrolyses by ÃŽÂ ²-galactosidase enzyme but not for IPTG molecules (Hans Noll Joseph Orlando, 1960). Hypothesis: a) IPTG activates beta-galactosidase enzyme at most effective effects. b) The rate of beta-galactosidase enzymatic activities depend on the time of induction. Materials and Methods: Part A: Time course of induction of ÃŽÂ ²-galactosidase by IPTG Induction of the ÃŽÂ ²-galactosidase enzyme. Two different sets of culture condition were investigated. One set in the condition with IPTG (5mM) and another set in the condition of adding water as the control experiment. 15 labeled microfuge tubes which contain 100  µl of the CTAB solution which used to kills the E. coli cells and lyses the cells to release the contents including ÃŽÂ ² -galactosidase were prepared and placed in the ice bath. 2.5ml of actively growing Escherichia Coli K12 was transferred into two separate 50ml conical flasks and covered with the foil immediately and then immersed in the temperature of 37  °C shaking water bath. 250 ÃŽÂ ¼l of water was added into the control flask and note the time as t=0 and then transferred 200 ÃŽÂ ¼l of the E. coli culture out immediately into the microfuge tube which labelled as 0c tube, mixed well and stored in the ice bath. The same procedure for another set of conical flask but 250 ÃŽÂ ¼l of IPTG was used inst ead of water. After that, two conical flasks were placed in the shaking water bath to maintain the temperature constant at 37 °C.The previous two steps were repeated for preparing the 1, 2, 3, 4, 5, 7, 10, 12, 15, 30 and 45 minute time points for the induction flask and 15 and 45 minutes time points for control flask. ÃŽÂ ²-galactosidase activity of each sample was ready to be observed after addition ONPG and Na2CO3 which used to stop the assay activity by changing the pH value to 11. (School of Biotechnology and Biomolecular Sciences, 2012). ÃŽÂ ²-galactosidase Assay. 15 sample of microfuge tubes were placed in the 37 °C water bath for 5 minutes to reach thermal equilibrium. Addition of 200 ÃŽÂ ¼l of 3mM ONPG into each sample at every 30 intervals and addition of 300 ÃŽÂ ¼l of 1M Na2CO3 into the microfuge tube followed by order after exactly 5 minutes of time of ONPG induction to deactivate the ÃŽÂ ²-galactosidase enzyme activities. The time of ONPG induction was recorded. All samples were centrifuged for 5 minutes and then 300 ÃŽÂ ¼l of supernatant of each sample was taken out and read the absorbance under 414nm with 300 ÃŽÂ ¼l water used as the blank. (School of Biotechnology and Biomolecular Sciences, 2012). Part B: Characteristics of the induction of ÃŽÂ ²-galactosidase Exactly same procedures in the Part A were carried out but several different of conditions were tested in this experiment following by: *A. 250 ÃŽÂ ¼l of IPTG (5 mM) and 250 ÃŽÂ ¼l of water (this is to keep the culture at close  to the same concentration for all alternatives) *B. 250 ÃŽÂ ¼l lactose (20 mM) + 250 ÃŽÂ ¼l H2O. *C. 250 ÃŽÂ ¼l IPTG (5 mM) + 250 ÃŽÂ ¼l glucose (20mM). *D. 250 ÃŽÂ ¼l IPTG (10 mM) + 250 ÃŽÂ ¼l glucose (20mM).. E. 250 ÃŽÂ ¼l IPTG (5 mM) then, after the 10 min sample is removed, add 250 ÃŽÂ ¼l  chloramphenicol (200ÃŽÂ ¼g/ml). F. 250 ÃŽÂ ¼l IPTG (5 mM) then, immediately after the 10 min. sample is removed,  add 250 ÃŽÂ ¼l rifampicin (250 ÃŽÂ ¼g/ml). G. 250 ÃŽÂ ¼l IPTG (5 mM) then, immediately after the 10 min. sample is removed,  add 250 ÃŽÂ ¼l streptomycin (500 ÃŽÂ ¼g/ml) . Note: At zero time point, all materials were added into set A, B, C and D while the antibiotic that used in the set E, F and G was added after 10 minutes time points sample has been taken out. (School of Biotechnology and Biomolecular Sciences, 2012). Discussions: From the graph shown in the figure 2, it can clearly see that the units of beta-galactosidase per ml of bacterial culture show a positive results when IPTG used in the induction but no response when water used instead of IPTG. The longer the IPTG induction time, the greater the units of beta-galactosidase per ml of bacterial culture produced. It can be explained that, IPTG acts as the inducer which depressed the repressor protein into inactive form by undergoes conformational change in the shape of the repressor protein that prevent them from binding to the operator region. Thus, the RNA polymerases can bind to the promoter site without any obstacles, transcription of lac operon occurs. Therefore, it can be concluded that inducer is playing a significant role in inducing of beta-galactosidase enzyme. Model data provided by the coordinator was used instead of the raw data because there is induction timing error when transferred the sample which causes the failure of the group results as it can notice that there is a sudden decrease in the value of the beta-galactosidase per ml of bacterial culture produced during 12 minutes in the figure 1. Based on the information provided in the part B experiment, the highest value of unit of beta-galactosidase produced was observed when IPTG was presence in the culture environment. An increasing trend of response with lower efficiency of effect were shown in the lactose, IPTG (5mM)+glucose and IPTG (10mM)+ glucose induction. However, as rifampicin, streptomycin and chloramphenicol added into the culture samples, a increasing concentration of beta-galactosidase enzyme at the beginning of experiment until 10 minutes then the reactions started to maintain at the constant level. The observations can be elucidated that when both IPTG and lactose were used as the inducer, they play the similar mechanism but the only reason that causes lactose had lower performance is IPTG will not be broken down during reaction whereas lactose will be degraded or used by the cells. As the rate of lactose degradation increases, the concentration of inducer in the culture decreases. In addition, glucose and galactose were formed after hydrolysis of lactose molecule. Glucose molecule is more preferred than the lactose molecule by the E.coli. Hence, a lower performance was shown when lactose was used as the inducer and IPTG always the best choice of inducer to use in the experiment. Since the glucose molecules involved in the experiment, a mechanism named as catabolite repression can be used to illustrated the other two IPTG (5mM)+glucose and IPTG (10mM)+ glucose conditions. Catabolite repression is a mechanism that represses the transcription process by introducing glucose molecules into the reaction since E.coli is more preferred glucose than IPTG while IPTG is essential for switching on the reaction. When the concentration of glucose molecule increases, the level of cyclic-AMP becomes lower. CAMP is required to start the transcription process as it is needed to binds with the Catabolite activator protein (CAP protein) and form an active complex which promotes RNA polymerases binds to promote region. Therefore, if cAMP level is low, there is inactive complex produced it unable to deactivate the repressor protein so repressor protein will bind to o-site and inhibit the occurrence of the transcription process. On the other hand, if the glucose molecule is absence, the high level of cAMP permit the transcription process to take place due to the cAMP binds to the CAP protein to form cAMP.CAP complex and deactivated the repressor protein, transcription takes place. Since the glucose is the preference substrate so when concentration of IPTG increases, it also will not affect the result when glucose is supplied. CTAB solution which also defined as the cetyl trimethyl ammonium bromide and used in the experiment to remove the E.coli cells and also destroyed the membrane of the E.coli cells in order to release ÃŽÂ ²-galactosidase enzyme that needed for the experiment from its content. E. coli is the source of the beta-galactosidase enzyme in the experiment. In the IPTG+ chloramphenicol culture condition, there was only IPTG inside the sample at the first 10 minutes and induction of enzyme was occurs but after Chloramphenicol was added the units of beta-galactosidase of bacterial culture remain constant due to the reason that Chloramphenicol is an antibiotic that inhibit the protein synthesis process and growth of E.coli (Ambrose,P.J,1984). The polypeptides synthesise of the RNA in the E.coli was hindered when added Rifampicin (Campbell, E. A et al, 2001). Rifampicin changes the shape and structure of the ribosomes which makes the lyses of the ribosomes (Sippel Hartmann, 1968) and also preven ts RNA polymerases from binding to promoter region. After streptomycin was added into the culture sample, Streptomycin inhibits growth of the E.coli by leading to misread the mRNA and protein synthesize disturbance when low amount of streptomycin provided (Modolell,  Juan, 1969). Hence, induction of beta-galactosidase activity was prohibited. Nevertheless, high quantity of Streptomycin added will even cause the death of E.coli.

What Leadership Means Essay -- Leadership Leaders Management Essays

What Leadership Means Leadership is 'the behaviour of an individual when he is directing the activities of a group towards a shared goal'. (Hemphill and Coons, 1957, p.7) A leader is interpreted as someone who sets direction in an effort and influences people to follow that direction. How they set that direction and influence people depends on a variety of factors. To really comprehend the 'territory' of leadership, one should briefly scan some of the major theories, notice various styles of leadership and review some of the suggested traits and characteristics that leaders should have. There are many leadership theories. Arthur G. Jago (1982) proposed a framework that organizes leadership theories based on each theory's focus and approach. "Focus" refers to whether leadership is viewed as a set of traits or as a set of actions. Focus on Traits: Theories with such a focus see leaders as having certain innate or inherent personality traits that distinguish them from non-leaders. These personality traits are supposed to be relatively stable and enduring. Focus on Behaviour: Theories with this type of focus see leadership as observable actions of the leader instead of personality traits. "Approach" is concerned with whether a particular theory or model of leadership takes a universal or a contingent perspective. Universal Approach: This approach believes that there is a universal formula of traits or behaviour for an effective leader. In other words, the universal approach assumes that there is "one best way" to lead in all situations. Contingent Approach: Contrary to the universal approach, the contingent approach does not believe the "one best way" formula. It believes that effective leadership depends on the specific situation. I am going to analyse two theories in detail, which according to me appear contradictory are Blake and Mouton?s Managerial Grid Theory (1978) under the head behaviour theories and Fiedler?s Contingency theories under the head contingencies theory. Behavioural Theories For over 20 years, a major thrust in leadership research has focused on the various behavioural patterns or styles used by different leaders and the functions fulfilled by these individuals. This research examined the impact that leadership behaviour had on the performance and satisfaction of followers. From... ... totally group or task-oriented; group members and the situation itself, all influence a leaders effectiveness. The leader needs to be aware of his own behaviour and influence on others, individual differences of group members, group characteristics, task structure, environmental and situational variables, and adjust his leadership style accordingly. Leadership needs to be adaptive. Organizations have changed over the past several decades with regard to the structuring of work methods and processes. Moving away from the traditional hierarchical design, most organizations are developing flatter, leaner structures that support a more empowered, team-based workforce. The nature of leadership has also changed significantly over time. References Gary A. Yukl, (2002) Leadership In Organizations, Prentice-Hall International Edition, 2nd Edition, Peter L. Wright, (1996) Managerial Leadership, Routledge, 1st Edition, Bittel L. R., The McGraw-Hill 36 ?hour Management Course, (McGraw-Hill, 1989), Fred E. Fiedler, (1987), A Theory Of Leadership Effectiveness, McGraw-Hill, 1st Edition Blake, R. R. and Mouton, J. S. (1978) The New Managerial Grid, Houston TX What Leadership Means Essay -- Leadership Leaders Management Essays What Leadership Means Leadership is 'the behaviour of an individual when he is directing the activities of a group towards a shared goal'. (Hemphill and Coons, 1957, p.7) A leader is interpreted as someone who sets direction in an effort and influences people to follow that direction. How they set that direction and influence people depends on a variety of factors. To really comprehend the 'territory' of leadership, one should briefly scan some of the major theories, notice various styles of leadership and review some of the suggested traits and characteristics that leaders should have. There are many leadership theories. Arthur G. Jago (1982) proposed a framework that organizes leadership theories based on each theory's focus and approach. "Focus" refers to whether leadership is viewed as a set of traits or as a set of actions. Focus on Traits: Theories with such a focus see leaders as having certain innate or inherent personality traits that distinguish them from non-leaders. These personality traits are supposed to be relatively stable and enduring. Focus on Behaviour: Theories with this type of focus see leadership as observable actions of the leader instead of personality traits. "Approach" is concerned with whether a particular theory or model of leadership takes a universal or a contingent perspective. Universal Approach: This approach believes that there is a universal formula of traits or behaviour for an effective leader. In other words, the universal approach assumes that there is "one best way" to lead in all situations. Contingent Approach: Contrary to the universal approach, the contingent approach does not believe the "one best way" formula. It believes that effective leadership depends on the specific situation. I am going to analyse two theories in detail, which according to me appear contradictory are Blake and Mouton?s Managerial Grid Theory (1978) under the head behaviour theories and Fiedler?s Contingency theories under the head contingencies theory. Behavioural Theories For over 20 years, a major thrust in leadership research has focused on the various behavioural patterns or styles used by different leaders and the functions fulfilled by these individuals. This research examined the impact that leadership behaviour had on the performance and satisfaction of followers. From... ... totally group or task-oriented; group members and the situation itself, all influence a leaders effectiveness. The leader needs to be aware of his own behaviour and influence on others, individual differences of group members, group characteristics, task structure, environmental and situational variables, and adjust his leadership style accordingly. Leadership needs to be adaptive. Organizations have changed over the past several decades with regard to the structuring of work methods and processes. Moving away from the traditional hierarchical design, most organizations are developing flatter, leaner structures that support a more empowered, team-based workforce. The nature of leadership has also changed significantly over time. References Gary A. Yukl, (2002) Leadership In Organizations, Prentice-Hall International Edition, 2nd Edition, Peter L. Wright, (1996) Managerial Leadership, Routledge, 1st Edition, Bittel L. R., The McGraw-Hill 36 ?hour Management Course, (McGraw-Hill, 1989), Fred E. Fiedler, (1987), A Theory Of Leadership Effectiveness, McGraw-Hill, 1st Edition Blake, R. R. and Mouton, J. S. (1978) The New Managerial Grid, Houston TX

Chapter 2 Review of Related Literature Sample

CHAPTER 2 REVIEW OF RELATED LITERATURE AND STUDIES FOREIGN STUDIES In its broadest sense, impeachment is the process by which public officials may be removed from office on the basis of their conduct. Strictly speaking, it is the decision by a legislature to accuse an official of one or more offenses that warrant removal according to constitutional standards. A vote to impeach then triggers a trial based on those charges. The most famous impeachment proceedings have involved presidents, but every state has its own procedures. Most follow the federal model in general, but vary widely in their specifics.At the federal level, impeachment starts in the House of Representatives, where members may initiate resolutions to impeach a sitting president. The House Judiciary Committee decides if a resolution merits a formal impeachment inquiry. A simple majority vote in the full House can launch a formal inquiry. The House Judiciary Committee conducts an investigation to determine if allegations against a president warrant charges, or articles of impeachment. If a simple majority of the full House votes to charge a president with at least one article of impeachment, that indictment will move to the Senate for trial.At that point, the president has been â€Å"impeached† by the House. House members act as or appoint congressional prosecutors. The chief justice of the Supreme Court presides over the trial in the Senate chamber. A two-thirds vote is required to convict and remove from office. The U. S. Constitution states that, â€Å"The President, Vice President and all civil Officers of the United States, shall be removed from Office on Impeachment for, and Conviction of, Treason, Bribery, or other high Crimes and Misdemeanours. † (Article II, Section  ). The House of Representatives has impeached two Presidents: Andrew Johnson and Bill Clinton.Johnson was charged in 1868 with eight articles of impeachment, but was acquitted by a single vote in the Senate tri al (depicted in the above engraving). Bill Clinton was charged with four articles of impeachment by the House in 1998, but was acquitted by the Senate early the next year. Richard Nixon resigned in 1974 before a final vote in the full House could send him to trial on three articles of impeachment. Each state constitution outlines a unique impeachment procedure, including variations on the list of impeachable offenses, protocol for an impeachment trial and the body responsible for an initial investigation.According to the Associated Press, seven governors in U. S. history have been removed from office following impeachment proceedings. The National Conference of State Legislatures said that a longer list would include states that have investigated governors for alleged offenses, voted to impeach a governor ahead of a trial, or held trials that resulted in acquittal. The only governor to be removed from office in the last 80 years was Gov.Evan Mecham of Arizona, who was convicted in 1 988 of obstructing justice and misusing $80,000 in state money that he was charged with funnelling to his car dealership to keep it afloat. In January 2008, the Illinois House of Representatives voted 114-1 to impeach Gov. Rod Blagojevich for abuse of power in connection with the federal investigation that had led to his arrest the month before. Mr. Blagojevich was charged with trying to sell the Senate seat vacated by Barack Obama and with seeking to extort campaign contributions in return for official actions, including providing reimbursement to a hospital.Following the process that has been generally adopted by state legislatures in recent decades, the Illinois House created a special investigative committee, which made a recommendation in favor of impeachment to the entire body. In all states except Alaska, Nebraska and Oregon, the House votes on articles of impeachment ahead of a trial. In Alaska, the process is reversed, according to The Book of States. That state's Senate mu st impeach a governor by voting on impeachment articles in order to initiate a trial in the House. Nebraska is the only state with a unicameral legislature.Without a state House, the Nebraska Senate votes to impeach before passing articles on to the state Supreme Court for a trial. Oregon is the only state without constitutional provisions for impeachment of a governor or other executive and judicial officers, according to the NCSL. Those officials may be removed from office, but not by the state's legislature. State courts in Oregon may try public officials for criminal offenses, but the procedure depends upon the jurisdiction of a crime. LOCAL STUDIES Section 1, Article XI of the 1987 Constitution declares that â€Å"Public office is a public trust.Public officers and employees must at all times be accountable to the people, serve them with utmost responsibility, integrity, loyalty, and efficiency, act with patriotism and justice, and  lead modest lives. â€Å"   These words echo loud and clear today as our country’s leaders find themselves at the brink of conducting this constitutional process. Impeachment has been defined as a national inquest into the conduct of public men. It is a necessary safeguard to ensure that public officers have the moral fitness and integrity to fulfil their mandate.The provisions on impeachment are enshrined in Article XI of the 1987 Constitution. Under the Constitution only the following public officers may be impeached: The President, Vice-President, the Members of the Supreme Court, the Members of the Constitutional Commissions, and the Ombudsman. This list of officers is exclusive. All other public officers and employees may be removed from office as provided by law, but not by impeachment. The grounds for impeachment are: culpable violation of the Constitution, treason, bribery, graft and corruption, other high crimes, or betrayal of public trust.These grounds are exclusive and offenses not falling within these parameters shall not be sufficient for impeachment purposes. The process begins at the House of Representatives, which has the exclusive power to initiate all cases of impeachment. A verified complaint must be filed by either a Member of the House of Representatives or by any citizen upon a resolution of endorsement by any Member thereof. Once the verified complaint has been filed it shall be included in the Order of Business within ten session days, and referred to the proper Committee within three session days thereafter.The Committee, after hearing, and by a majority vote of all its Members, shall submit its report to the House within sixty session days from such referral, together with the corresponding resolution. The resolution shall be calendared for consideration by the House within ten session days from receipt thereof. In the committee hearings, a vote of at least one-third of all the Members of the House shall be necessary either to affirm a favourable resolution with th e Articles of Impeachment of the Committee, or override its contrary resolution.The vote of each Member shall be recorded. If however, the verified complaint or resolution of impeachment is filed by at least one-third of all the Members of the House, the same shall constitute the Articles of Impeachment, and trial by the Senate shall forthwith proceed. The Senate has the sole power of sole power to try and decide all cases of impeachment. When sitting for that purpose, the Senators shall be on oath or affirmation. When the President of the Philippines is on trial, the Chief Justice of the Supreme Court shall preside, but shall not vote.No person shall be convicted without the concurrence of two-thirds of all the Members of the Senate. The person impeached shall be  removed from office  and shall be  disqualified to hold any office under the Republic of the Philippines, but the party convicted shall nevertheless be liable and subject to prosecution, trial, and punishment accord ing to law. It is clear that the liability does not end at the Senate, the person impeached shall also be held for appropriate action as a result of his illegal and improper acts.

To Kill a Mockingbird Movie vs Novel - 1238 Words

What You See Is Not What You Read Life is full of lessons, the movie version of the novel To Kill a Mockingbird by Harper Lee is not! Harper Lee entwines these life lessons throughout her novel; however, the movie version fails to incorporate these lessons into its plot. Thus, the movie version must be deemed incomplete, as it is most certain that there are many important lessons that Jem and Scout learn throughout the novel. A few key characters who taught these lessons to the children and who will be the subject of this essay are Ms.Dubose and the morals learnt from her successful fight against morphine addiction, the role of Aunt Alexandra in front of Scout as a female role model and lastly Mr.Dolphus Raymond’s preferred associations†¦show more content†¦This relationship forms on the other side as well. Scout begins to understand what being a lady is about, at one point she say, â€Å"After all, if Aunty could be a lady at a time like this, so could I.†(Lee 318). Throughout the book Aunt Alexandra’s views and actions become less ignorant and Scout becomes more understanding about what Aunt Alexandra has to say. It is a pity that the director refrained from including Aunt Alexandra in the movie version. This was most probably done because Aunt Alexandra has something to say on almost everything that happens and therefore there would have been a lot more scenes in the movie if she were included. However, it was indeed a bad choice because the character of Aunt Alexandra would have made the movie much more interesting. Thus, the movie must be deemed incomplete compared to the novel. Another non-prejudice character who was not included in the movie was Mr.Dolphus Raymond. He teaches Scout and Dill a very important lesson that reverberates throughout the novel; All that is gold does not glitter (J. R. R. Tolkien). 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