Hang ToBIOL 2013-Fall 2018Beta-galactosidase post-labAnalysis of Induction of Lac Operon in E.
coli by IPTG and ONPG, and Comparison of How Beta-galactosidase is Produced with Presence of LactoseINTRODUCTIONEscherichia coli (E. coli), rod shaped bacteria and found in a lower intestinal tract of human, is an important model organism in biology. Although E.
coli is known as a cause of some poisoning food due to the strain 0157:H7, most kinds of E. coli bacteria do not cause disease in human. E. coli was the first organism to have its complete genome sequenced in 1997(5).
E. coli has long been used in biology and genetics due to their ease of genetic manipulation. The regulation of genes, which consists of lac operon, controls the metabolism of lactose in the bacteria E. coli. Lac operon contains genes and two important regions which are promoter and operator regions. Promoter region is a place where the transcription process initiates if only RNA polymerase can bind to, while the operator region is a place where repressor binds to and stimulates the genes to turning on or off the transcription process.The structure of operon consists of three important genes: lacZ, lacY and lacA(3).
LacZ gene encodes the enzyme Beta-galactosidase, which hydrolyzes Beta-galactosides, such as lactose, into monosacccharides. A frameshift mutation in the lacZ gene can affect the function of the lac operon, resulting in nonfunctioning protein of beta galactosidase. This in turn will make the lac operon non-inducible and metabolism will not occur. Galactoside acetyltransferase is encoded by lacA, while lactose permease is required to transport lactose molecules and is encoded by lacY(2).Genes can be regulated positively or negatively(2). When repressor is not bound to operator cAMP-CAP complex can bind to promoter, which indicates that the lac operon transcription happens. Glucose and cAMP levels are controversy each other. When the amount of glucose is high cAMP level is low, thus the complex of cAMP-CAP cannot produce to make the transcription continue.
The complex of cAMP-CAP is only produced when the amount of cAMP is high and in levels of low glucose. The formation of the complex allows RNA polymerase to bind to promoter and make transcription occur(1) . This mechanism is called positive control(2). Within an inducer, like isopropyl beta-D-1-thiogalactopyranoside (IPTG) repressor cannot bind to the operator, which results in continuance of transcription due to binding of RNA polymerase to the promoter(1).
Without an inducer, repressor is bound to operator and RNA polymerase can bind to the operator, hence transcription is inhibited. This is called negative control (2).Ortho-Nitrophenyl-galactosidase (ONPG) is a substrate and is used to measure beta-galactosidase activity. ONPG is cleaved like lactose.
Both lactose and ONPG are colorless, which is hard to examine; ONPG yet can obtain yellow color due to beta-galactosidase hydrolyzation when quantified by using a Spectrophotometer.The study focuses on these things. Firstly, a minimum concentration of IPTG is necessary for the fullest expression of Beta-galactosidase. On the other hand, glucose causes a decrease in lacZ activity.
Although there is a maximum concentration of IPTG, the expression of Beta-galactosidase still cannot be in full. Secondly, the Beta-galactosidase enzymatic activity increases with time after induction.MATERIALS AND METHODSGene expression, typically ?-galactosidase enzyme assay, was observed and recorded to see how a single promoter element would affect it under a variety of conditions.Experiment 1:The purpose of experiment #1 was to quantify differences in the induction of Beta-galatosidase expression over time. Cultures for E. coli strains were lacZ- and lacZ+, grown at 37?C and induced with IPTG at Time zero. Two ml of each culture was transferred into two tubes, 1ml for each tube, labeled tube A (control), and tube B (the experiment) to which 0.2ml of 0.
013M ONPG was added. These tubes, 4 tubes in total, were then incubated in 20 minutes at 37?C. To stop the reaction, 2.7ml of 1M Na2CO3 was added to all four tubes. One ml of each tube was later transferred into a cuvette to obtain the absorbance at 420nm for solutions of tube B, while the contents of the tube A were used as blank. These steps were repeated over time at 30, 60 and 90 minutes. The total amount of ONP produced (mmol) is determined by dividing the absorbance by 0.004.
The activity of Beta-galactosidase is calculated by dividing the amount of ONP produced by 20.Experiment 2:This experiment was performed to see how inducers and repressors would affect beta-galactosidase expression. Part 1 would test how IPTG would induce expression of gene.
There were 6 cultures of lacZ+ E.coli with the following amounts of IPTG or glucose: nothing, 1 µM, 10 µM, 100 µM, 10mM glucose, and 100 µM IPTG and 10mM glucose were added into tube 1, 2, 3, 4, 5, and 6, respectively. Tube 1 was control. Part 2 would determine how gene expression would react with the presence of glucose in tube 5 and 6.
Beta-galactosidase activity of each culture was measured using standard protocol as experiment #1.Experiment 3:Wild-type E. coli and mutant strains which were used in this experiment would reveal the genotypes of four unknown strains. There were four strains: CA800 was wild type and functional; 30SO could not make ?-galactosidase enzyme due to a frameshift mutation; CA7089 had a mutation in operator site, which made it constitutive; YA694 was a “superrepressor” mutation, and unable to regulate expression although it could make a very small amount of enzyme and was functional. These were 2 streaked plates: one was the LB/X-gal plate and another plate was same in addition to IPTG. Each of four unknown strains would be transferred into an appropriate position marked as 1 to 4 on the plates, respectively by streaking to spread the bacteria.
A toothpick must be sterilized every time using. Both plates were then labeled and incubated at 37?C in 16~18 hours. The color of colonies on X-gal plates would determine whether lac operon would be constitutive, inducible or unexpressed in that strain.
It would be lacZ+ if colonies turned blue with the presence of IPTG, while lacZ- colonies would remain white on both plates. In lacIs colonies would be colorless, and mutation would be supperrepressed. Colonies on plates with lacOc genotype would have the darkest blue; lacZ expression would be constitutive. RESULTS AND DISCUSSIONSExperiment 1In experiment one, the results of the assays showed little activity in the lacZ+ and lacZ- cultures over time on the graph 1. In the lacZ-, there was likely a straight line at units 0 of activity meaning that no beta-galactosidase was made at all. Although at the 60-minute, it was 0.025 units, while it was concluded 0 values at the time 0, 30, and 90 due to the negative values of absorbance reading.
This did not really affect to the final result of the lacZ- culture, which was a straight line without the up or down slope at all the time points. At the zero time, activity for lacZ- was zero, while it was 0.55 units in the lacZ+.
In the lacZ+ culture, all units of activity were not zero and were tending increasing meaning that the inducer, IPTG, increases the ?-galactosidase activity over time as expected and supported the hypothesis. Typically, in the lacZ+, there was a steadier rate seen at the 30 and 60 minutes time points, which increased from 7.85 to 23.9 units. However, there was a downslope at unit 11.7 when the culture reached to the 90-minute time point. The unit values at the time 30, 60 and 90 were higher than expected ones, because this came from an error in the experiment.
Some dilutions such as 4-folds (at the time 30 and 90 minutes in the lacZ+) and 2-fold (at the time 60 minute in the lacZ+) were processed due to high values of absorbance reading (greater than 1). It was supposed to be an increasing slope since the beta-galactosidase would increase when increasing the time for the lac operon to be induced by IPTG. What accounting for these differences might come from human error. Upon the results, it was concluded that the more time that the lac operon is allowed to be induced by IPTG, the more activity there will be. These results supported the initial hypothesis.Experiment 2In experiment two, the increase of IPTG improved lacZ activity. Typically, on the graph 2, there was an increase of lacZ activity, on the samples 1 and 2, from 0.
313 to 0.367 units when the amount of IPTG was increased from 1µM to 10 µM. The affection of the increase of the IPTG was also observed when increasing the IPTG from 10 µM to 100 µM; as a result, there were 0.367 units on the sample 2, while there were 0.495 units on the sample 3. These observations supported the hypothesis that an increase in a promoter inducer, which in this case was IPTG, would cause an increase in lacZ induction. On the other hand, the addition of glucose saw a decrease in lacZ activity. For instance, on the sample 4 the activity was reduced from 0.
495 units (on the sample 3) to 0.35 units. Upon to the sample 5, the activity had a rise up to 1.138 units when adding 10 mM glucose to 100µM. This unit here on the sample 5 was supposed to lower than one on the sample 4, since the small amount of glucose added to it.
This difference might come from human errors using the machinery as well. These numbers here were not 100% correct most likely due to some human error, however there was the overall trends of increase activity with inducer and decreased with a repressor (except for the sample 5). With those facts it could be deduced that inducer concentration and gene expression are proportional.Experiment 3It was given that there were one wild-type and three mutants expressed on each of the two plates. Graph 3 showed that the genotypes of four unknown strains were revealed by matching the plates to the mutations in presence or absence of the inducer, IPTG.
The strain 1 had no blue meaning that there was no growth so it was 30SO, the lacZ-, because this mutant could not produce the beta-galactosidase enzyme at all. The strain 2 was a little strange since blue was on both plates. It was supposed that blue would only show up on the plate of X-gal with ITPG rather than on the plate of X-gal due to the presence of IPTG. Again, this difference might come from human error while doing this experiment. The strain 3, there was blue on the X-gal plate, while it was white showed on X-gal which added IPTG.
It had to be YA694, the lacIS, since mutant in the lacI gene was able to prevent repressor from binding in inducer which made the lacZ gene have not functioned. A very small amount of beta-galactosidase was in the cell, which turned the colonies into a very slightly blue on the X-gal plate. On the other hand, there would be no color in the presence or response of the inducer. The strain 4 had to be the CA7089 because it had a mutation on the operator which presented the repressor from binding to the operator site and made the gene expression constitutive. Overall conclusion and comparison to published literature.This study helps to confirm what has already been known. Concentration of IPTG affects the expression of Beta-galactosidase in different ways. The enzyme can be expressed in the highest values when the maximum of concentration of IPTG added, or it can be the lowest values in a minimum concentration of IPTG.
Glucose is a substrate that reduces expression of Beta-galactosidase. The higher glucose is, the lower Beta-galactosidase expresses. This study can also help to genotype four unknown strains, which are lacZ+, lacZ-, lacOC and lacI. LacZ+ is wild type, which means blue dye can be observed, while lacZ- cannot. LacOC is constitutive, thus Xgal turns the colony blue in the most expression; however, lacIS inhibits plate to turn onto blue even though there is the presence of inducer. Beside which has already known, this research clears out how it does not completely support to published data. On this experiment to check lacZ activity with inducer over time, the Beta-galactosidase enzymatic activity increased with time after induction. Typically, lacZ expression increased from 0.
55, 7.85 to 23.9 units at 0, 30 and 60 minutes when inducer added. This work supports experiment of Jacob and Monod, which was done in 1960.
In their experiment, the enzyme assay trended to increase after induction from 0.5, 3.6, 11 to 14.3 units at 0, 2, 4, and 5 hours(3) . The enzyme activity in their study still increased up to 16 units at 6 hours, while this study had a decrease to 11.
7 units at 90 minutes. This can explain by human error during experiment. Overall, the activity of Beta-galactosidase enzyme increases with time after induction.
In future, it is essential to use lab equipment precisely and follow required procedure to be able to get the result as expectation and limit unnecessary error.ReferenceCampbell, M. & Farrell, S. 2009, “Transcription Regulation in Prokaryotes” in Biochemistry, ed.
A., 6th edn, pp. 296Jacob, François, and Jacques Monod. “Genetic Regulatory Mechanisms in the Synthesis of Proteins.” Journal of Molecular Biology, vol. 3, no. 3, 1961, pp. 318–356.
Jacob, François, and Jacques Monod. “Genetic Regulatory Mechanisms in the Synthesis of Proteins.” Journal of Molecular Biology, vol. 3, no.
3, 1961, pp. 332.Murray, V. 2012, “Induction of Beta-galactosidase in Escherichia coli” in Principles of Molecular Biology (Advanced), pp. 51″Online Education Kit: 1997: E. Coli Genome Sequenced.” National Human Genome Research Institute (NHGRI), May 2013DATAGraph 1: graph of LacZ activity over timeTime point (minutes) Activity (units)0 030 060 0.02590 00 0.
5530 7.8560 23.990 11.7Graph 2: LacZ induction by IPTG and glucoseSamples Activity(units)1 0.3132 0.3673 0.4954 0.
355 1.138Graph 3: Results of Predicting the Genotypes of Four lac strains789940-45148500Growth medium Strain 1 2 3 4X-gal (1)- + + +++X-gal+IPTG (2)- ++ – +++Predict the genotype: LacZ- LacZ+ LacIsLacOcJustify your rationale. Why would you see this result with the hypothesized mutant?The lacZ- in both plates was white meaning no blue color after incubation because ?-galactosidase could not be made even if an inducer is there or not.
Both plates turned a blue color: a darker blue plate(2) and a light blue on the plate(1). The expression of lacZ was significantly higher in the presence of this inducer than the absence of an inducer. The lacIs suppressed the promoter and could not function. ?- galactosidase was expressed as no color on the plate(2), while colonies on the plate(1) turned to slightly blue since there were a few molecules of ?-galactosidase in the cell. Both colonies turned dark blue because the repressor could not bind. As a result, the operator stopped the repressor from binding to the operator region, and lacZ gene was expressed constitutively.Plate A: X-gal with IPTGPlate B: X-galPlate C: X-gal (to the left) and X-gal with IPTG (to the right)