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Aim: To study the effects of IPTG, lactose, glucose, chloramphenicol, rifampicin and streptomycin induction on the units of beta-galactosidase of E.coli
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 Î²-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 Î²-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 Î²-galactoside permease which playing the role in transporting lactose molecules into the cell while lacA is converted into Î²-galactoside transacetylase through transcription process which is an enzyme that involved in adding an acetyl group (CH3) from acetyl coenzyme A to the 6′ position of the Î²-galactosidase (Xing Guo, Wang, Laurence R. & OlsenSteven L. Roderick, 2002). As the inducer such as allolactose (natural inducer) or IPTG, Isopropyl Î²-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 – Î²-galactosidase
ortho-Nitrophenyl-Î²-galactosidase which known as ONPG is used to measure the Î²-galactosidase enzymatic activities in this experiment which show a yellow colour when Î²-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 Î²-galactosidase enzyme but not for IPTG molecules (Hans Noll & Joseph Orlando, 1960).
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 Î²-galactosidase by IPTG
Induction of the Î²-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 Î² -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 Î¼l of water was added into the control flask and note the time as t=0 and then transferred 200 Î¼l 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 Î¼l of IPTG was used instead 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. Î²-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).
Î²-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 Î¼l of 3mM ONPG into each sample at every 30 intervals and addition of 300 Î¼l of 1M Na2CO3 into the microfuge tube followed by order after exactly 5 minutes of time of ONPG induction to deactivate the Î²-galactosidase enzyme activities. The time of ONPG induction was recorded. All samples were centrifuged for 5 minutes and then 300 Î¼l of supernatant of each sample was taken out and read the absorbance under 414nm with 300 Î¼l water used as the blank.
(School of Biotechnology and Biomolecular Sciences, 2012).
Part B: Characteristics of the induction of Î²-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 Î¼l of IPTG (5 mM) and 250 Î¼l of water (this is to keep the culture at close to the same concentration for all alternatives)
*B. 250 Î¼l lactose (20 mM) + 250 Î¼l H2O.
*C. 250 Î¼l IPTG (5 mM) + 250 Î¼l glucose (20mM).
*D. 250 Î¼l IPTG (10 mM) + 250 Î¼l glucose (20mM)..
E. 250 Î¼l IPTG (5 mM) then, after the 10 min sample is removed, add 250 Î¼l chloramphenicol (200Î¼g/ml).
F. 250 Î¼l IPTG (5 mM) then, immediately after the 10 min. sample is removed, add 250 Î¼l rifampicin (250 Î¼g/ml).
G. 250 Î¼l IPTG (5 mM) then, immediately after the 10 min. sample is removed, add 250 Î¼l streptomycin (500 Î¼g/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).
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 Î²-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 prevents 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.
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