Catabolite Repression and Induction of Beta-galactosidase Synthesis - Lab Report Example

Lab Report on Catabolite Repression and Induction of Beta-galactosidase Synthesis in E. coli Abstract The major aim for the experiment is to demonstrate both catabolite repression and induction of beta-galactosidase synthesis during growth of E. coli. Synthesis of the enzyme beta-galactosidase is induced in wild type E. coli strains in response to the presence of lactose, the enzyme's natural substrate. Apart from induction, synthesis rate is determined by catabolite repression, whereby it slows down the synthesis of beta-galactosidase especially in the presence of a better carbon (and energy) source, such as glucose. In this experiment, Escherichia coli (E. coli) is used as the bacteria to induce synthesis of enzyme β-galactosidase. The results support lactose metabolism by newly synthesised beta-galactosidase and also, quantitatively, IPTG is a more effective inducer of beta-galactosidase synthesis than lactose. Introduction In order to regulate the gene expression levels in a cell, there are certain mechanisms that must be considered in operation. In which case, the regulation are considered at transcription and translationt levels or the stability of messenger RNA. The aforementioned can only work in regulation based on the synthesis of a particular protein. Consequently, it comes out as a subject of importance to investigate the regulation of transcription of bacterial genes. For this case, Escherichia coli (E. coli) is used as the bacteria to induce synthesis of enzyme β-galactosidase. Escherichia coli (E. coli) can produce the enzyme β-galactosidase which breaks lactose into galactose and glucose. Synthesis of the enzyme beta-galactosidase is induced in wild type E. coli strains in response to the presence of lactose, the enzyme's natural substrate (Ring, 1999, 80). The inducer, lactose, is usually the molecule broken down by the enzyme system. Worth noting is the ability of E.coli to solely use lactose as a carbon source regardless of the presence of glucose. A more stable inducer that lactose, IPTG (Isopropyl β-D-1-thiogalactopyranoside) helps in inducing expression of the enzyme without being metabolized in the process. Apart from induction, synthesis of β-galactosidase is also influenced by catabolite repression. The process involves slow down of the synthesis process, facilitated by lactose, especially when presence of glucose is detected. Intuitively, glucose acts as a better energy and carbon source than lactose (Wallenfels, 1972, 67). When given both sugars, E.coli will not synthesize beta-galactosidase until all of the glucose is first exhausted from the medium. This experiment is designed to demonstrate both catabolite repression and induction of beta-galactosidase synthesis during growth of E. coli. Method Procedure There was removal of 5 ml aliquots from both control (C) and lactose (L) cultures with 1 ml aliquot of Lactorse culture placed in each plastic centrifuge tubes. 4 ml culture medium was added to make 5 from the obtained 1 ml aliquot. The absorbance for each case was read through the help of a culture medium at 620 nm. Further, chilling of the samples was done for 2 minutes and then centrifuged at 3500 rpm for 10 minutes followed by pouring the supertanant and using the waste bottle to discard. 2 ml of Tris HCl buffer was obtained and was added followeed by resuspending by pipetting up and down until a uniform suspension was achieved. Fresh labelled test tube was obtained and used to contain a fresh 1 ml aliquot of the bacterial suspension. 2 drop of toluene and vortex was then added within a span of 30 seconds. The enzyme was then placed in a shaking water bath to achieve liberation of the same. 0.5 ml of 0.005 M 0-nitrophenyl-beta-D-galactoside (ONPG) was added followed by incubating at 370C for 15 minutes. 5.0 ml of triethanolamine acetate (1.0M, pH 8.8) was added to stop the incubation process. There was transfer of the tube’s content into a centrifuge tube followed by centrifugin at 3500 rpm for 10 min. The absorbance of the supernatant at 420 nm was read against distilled water and the results recorded. Results The following table depict the results and calculations for cell density for cell density of the control and induced. The table shows that at 180 min, the cell density for the two, induced (1/5*) and control are equal. However, the situation differs after 30 minutes of cell growth whereby the induced exhibits a higher cell density as compared to the control Table 1: Table comparing cell density for control and induced Time (min)  A620 Cell Density (mg/ ml)   Control (c) Induced Induced Control Induced Induced (I) 1/5 1/5* 180 0.2 0.19 0.04 0.940*0.2 = 0.940*0.19 = 5*0.940*0.04 = 0.188 0.1786 0.188 210 0.22 0.2 0.05 0.940*0.22 = 0.940*0.2 = 5*0.940*0.05 = 0.2068 0.188 0.235 Note: The induced was multiplied by 1/5 to help in giving a more accurate results to help in covering for deviations resulting from dilution. For Table 2, - Galactosidase Activity, was examined. The table shows that the specific activity for - Galactosidase is much higher than for induced at both 180 minutes and 210 minutes into the experiment. Further, after 30 minutes interval from the 180th minute, the control witnessed a decrease in specific activity indifferece to the induced which exhibited an increase. Table 2: Table compairing - Galactosidase Activity for control and induced Time (mi) Activity (Unit/ ml) Specific activity (Unit/ mg cells) Control (c) Induced (I) Induced 1/5 Control (c) Induced (I) Induced 1/5* Control (c) Induced (I) 180 0.08 1.3 0.61 0.356 5.778 13.56 =1.894 13.56/ 0.1786 = 75.92 210 0.07 1.35 0.95 0.311 6.00 21.11 0.311/ 0.207 =0.05 21.11/0.188 = 112.3 specific activity for (i)= activity of induced (1/5*)/ cell density of induced The table 3 below shows a representation of mean and standard error calculated for each treatment which allowed for obtaining graph for mean specifc activity vs time. Table 3: A simple table made for comparing calculated means and SE for control and induced: Time (min) mean specific activity (unit/mg cells) standard error of the mean   control induced control (Series 1) induced (series 2) 0 5.39 3.77 8.256262 4.387083967 30 0.80 0.67 0.693635 0.592509633 60 0.93 10.22 0.272437 3.408459143 90 3.96 6.44 5.114627 4.550646317 120 1.57 61.39 0.218108 23.597247 150 3.84 44.53 5.008857 31.04544162 180 1.46 54.19 0.431708 31.04544162 210 1.35 44.05 0.261949 19.20573469 240 1.41 232.20 0.333748 129.3774269 From the table above, a graph was plotted as shown below. The graphs shows a generalt increase in specific activity of b-galactosidase with frequent minima and maxima point for the induced. However, for the control the situation is different as its shows low specific activity for beta-galactosidase and with insignificant change in the progress. Figure 1: Graph for mean specifc activity vs time after induction for beta-galactosidase activity Key: series 1 = control Series 2 = induced Figure 2: Graph with mean error bars Discussion Table 1 indicates that cell growth witnessed int the presence of lactorse was more as compared to that of the control. Usually, the activity of beta-galactosidase is measured in terms of cell growth: hence more cell density witnessed indicates that lactose is responsible for the the synthesis of beta-galactosidase in induced (I) (Lee, 2006, 45). However, for control the action of lactose was kind of repressed; hence the growth was slow leading to lower cell density. Table 2 shows that the specific activity for - Galactosidase is much higher than for induced at both 180 minutes and 210 minutes into the experiment. Further, after 30 minutes interval from the 180th minute, the control witnessed a decrease in specific activity indifferece to the induced which exhibited an increase. This also insinuates that the addition of lactose, for induced (I), was responsible for the production of beta-galactosidase hence the more specific activity than that observed for the control (Krivtsov & Armstrong, 2007, 65). For the graph shown above, the addition of lactose for induced (I) lead to general increase in specific activity over time accompanied with repeated maxima and minima point. This represent a different case to the control, whereby there was no defined trend with the graph showing insignificant change in the activity of the beta-galactosidace (Hall, 1997, 108). For the induced, after each 30 interval, from 0, there was a decrease in activity followed by an increase and then last interval witnessed a steep increase. From the results when lactose are present a decline is observed immediately after each lactose addition. A better explanation of this is that the free beta-galactosidase is utilized in metabolizing the available lactose into glucose and galactose at the start of the experiment (Dugdale, 2010, 56). Therefore, a decrease in beta-galactosidase activity is observed in these areas because most of it is used in the metabolism action and the presence of glucose inhibits action of lactose. A steep increase is then observed because of the addition of lactose, which facilitates thet synthesis of beta-galactosidase thereby leading to increase in activity. This cycle continues as the the time progress with maxima and minima points as witnessed in graph. However, as time progresses the level of glucose produced becomes low thereby increasing the action of lactose in synthesizing beta-galactosidase (Fowler, 2000). The graph indicates a plateau phase, which is accompanied by a slight increase and decrease in the specific activity of the enzyme. For all the experiments, lactose was shown to be responsible for the synthesis of the beta-galactosidase. In all these experiments lactose stimulated the production of newly synthesised beta-galactosidase, which lead to the increased specific activity observed. The absence of stimulant, such as IPTG (Isopropyl β-D-1-thiogalactopyranoside), lead to the occurrence of plateau phase during the metabolism of lactose. (Jacobson et al, 1994, 98). Consequently, this confirms that IPTG is a more stable inducer in expression of beta-galactosidase than lactose (Karp, 2005, 156). Conclusion The experiment was successful in meeting the aim as it was able to explore transcription of bacterial genes through exploring the activity of lactose in synthesis of beta-galactosidase. In all the experiments performed lactose was responsible for synthesis of beta-galactosidase. However, the lag phases for the activity of beta-galactosidase show the metabolism of lactose and the instability of lactose thereby calling for the need of IPTG, which is always stable. References Jacobson RH, Zhang XJ, DuBose RF, Matthews BW (1994) Three-dimensional structure of β- galactosidase from E. coli. Nature. 369:761-6. Karp G (2005) Cell and Molecular Biology Concepts and Experiments, 4th edition, John Wiley & Sons, Inc.,Hoboken. Ring M, Huber RE (1990) Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli). Arch Biochem Biophys. 283:342-50. Wallenfels K, Weil R (1972) “β-galactosidase” In The Enzymes (Boyer PD, ed), Vol VII, 3rd ed, Academic Press, New York, pp 617-663. Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (April 2006). "Senescence-associated beta-galactosidase is lysosomal beta- galactosidase". Aging Cell 5 (2): 187–95. doi:10.1111/j.1474-9726.2006.00199.x. PMID 16626397. Dugdale, M.L. et al. (2010). "Importance of Arg-599 of b-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop". Biochem.Cell Biol. (88): 969–979. doi:10.2210/pdb3muy/pdb. rendered with PyMOL Fowler AV, Zabin I (October 2000). "The amino acid sequence of beta galactosidase. I. Isolation and composition of tryptic peptides". J. Biol. Chem. 245 (19): 5032–41. PMID 4918568. Krivtsov, A. V., & Armstrong, S. A. (2007). MLL translocations, histone modifications and leukaemia stem-cell development. Nature Reviews Cancer, 7(11), 823-833. Hall, B. G. (1999). "Number of mutations required to evolve a new lactase function in Escherichia coli". Journal of bacteriology 129 (1): 540–3. PMC 234956. PMID 318653. Hall, B. G. (1981). "Changes in the substrate specificities of an enzyme during directed evolution of new functions". Biochemistry 20 (14): 4042–9. doi:10.1021/bi00517a015. PMID 6793063 Read More