Investigation of Induction Time of lac operon in E. Coli with IPTG, and Comparision of the Amount Beta-galactosidase produced with Lactose, IPTG and Antibiotic.
An operon is a group of genes that are arranged side by side with a regulatory gene. It also includes a promoter gene, operator gene, structural genes. Regulatory genes control transcription with positive or negative signal. (Jacob, and Monod, 1961) A positive signal, inducer, would stimulate binding of RNA polymerase by binding to the operator gene and transcription would occur. A negative signal, repressor, would not have any transcription occurring as it would not allow RNA polymerase to bind to promoter. This control of gene expression allows organism to not waste energy producing enzymes not needed. (Murray, 2012)
The lac operon has a promoter, operator, regulatory and structural genes. The structural genes consists of the lacZ, lacY and lacA gene which codes for enzymes beta-galactosidase, permease and thiogalactoside transacetylase respectively. (Campbell and Farrell, 2009) Lactose is dissacharide with galactose and glucose binded together by a glycosidic linkage. Beta-galactosidase hydrolyses that linkage, hence breaks down lactose to be used as a carbon source. (Campbell et al, 2009) Permease is a membrane-transport protein, it goes against the membrane potential concentration gradient to drive lactose into cell. (Kaback, Sahin-Toth and Weinglass, 2001) The function of thiogalactoside transacetylase is still being studied but there seems to be a connection defending the cell against antibiotics. (Andrews and Lin, 1976) The regulatory gene is called the lacI gene, it produces the repressor which causes the negative control for gene expression. (Murray, 2012)
The lac operon transcription occurs only when there is no repressor bound to the operator and when the cAMP.CAP complex is bound to the promoter. In levels of high glucose, cyclic AMP (cAMP) levels are low hence no cAMP.CAP complex produce to continue transcription. In absence of glucose, cAMP levels are high, this results in formation of cAMP.CAP complexes that binds to promoter region and attracts RNA polymerase to bind to promoter for transcription. (Campbell et al, 2009) This control mechanism is called catabolite repression and is a positive control. (Murray, 2012)
In presence of an inducer such as allolactose or isopropyl beta-D-1-thiogalactopyranoside(IPTG), the repressor and inducer binds together and becomes inactive and cannot be bound to operator anymore. (Campbell et al, 2009) Without any repressor bound RNA polymerase can continue with transcription of genes. The allolactose is a derivative of lactose and IPTG is a synthetic analogue. (Galea and Murray, 2012) Without an inducer, repressor is bound to operator and blocks transcription from occurring. This control is a negative one by the repressor. (Murray, 2012)
The binding RNA polymerase to promoter is weak so not only must there be no repressor blocking the promoter region, the cAMP.CAP complex must be bound to promoter for transcription to occur. (Campbell et al, 2009) In presence of glucose and in absence of inducer, no transcription would occur. (Murray, 2012)
Besides controlling of gene expression, antibiotics can be used to inhibit either transcription or translation. In this experiment, chloramphenicol, rifampicin and streptomycin are used. Chloramphenicol and streptomycin inhibits ribosome function, no protein synthesis can occur as it is inhibiting at the translation phase. (Neu and Gootz, 1996) Rifamicin inhibits DNA directed DNA polymerase, no transcription can occur. (Neu et al, 1996) The effects of inhibition at translation or transcription are observed.
In this experiment, induction time of lac operon will be determined by incubating Escherichia coli(E.coli) and IPTG for different time points and assaying its beta-galactosidase activity. IPTG is used here instead of allolactose as it will not be broken down by beta-galactosidase. Ortho-nitrophenyl-beta-galactosidase(ONPG) is used for the beta-galactosidase assay as a substitute for lactose substrate. O-nitrophenol is produced, it is yellow in colour. The assay is measured spectrophotometrically at an absorption of 414nm. The absorption reading is directly proportional to the activity of beta-galatosidase hydrolysing ONPG to o-nitrophenol. Also, induction of beta-galatosidase are compared by using lactose or addition of glucose or antibiotics such as chloramphenicol, rifampicin and streptomycin.
Haworth Structure of IPTG and ONPG is shown below.
Figure 1. Figure 2.
Figure 1 shows IPTG and Figure 2 shows ONPG.
Materials and Methods:
Refer to BIOC2201 Principles of Molecular Biology – Advanced Laboratory Manual, page 51-61 for material and methods for the experiment. For part B, Lactose was used as inducer, the rest of the results were obtained from the class.
There are 2 parts to this experiment. For part A, the induction time for beta-galactosidase in E.coli is determined. Water is used as a control. The beta-galactosidase per ml of bacterial culture is calculated by absorbance reading/ (absorbance coefficient of o-nitrophenol (21300M/cm)* path length(0.9cm)) multiply by assay volume(0.0008µl , (Galea et al, 2012) the induction time is determined when beta-galactosidase per ml of bacterial culture is significantly increasing with time. For part B, data for the lactose experiment and a compilation of class results are included. The calculations for beta-galactosidase per ml of bacteria culture is the same as part A.
Table 1 shows results for part A. with absorbance readings and corrected absorbance readings. The units of beta-galactosidase per ml of bacterial culture is included too. Calculations for it was explained earlier.
Graph 1 shows the units of beta-galactosidase per ml of bacterial culture for IPTG and water against time. From the graph, induction time is approximately 13-15mins.
Graph 3 shows the graph of units of beta-galactosidase per ml of bacterial culture against time for the experiment carried out using lactose as inducer.
Cetyl trimethyl ammonium bromide(CTAB) was used to kill the E.coli cells and release beta-galactosidase that have been produced after incubation. It is used so that the beta galactosidase can then be reacted with ONPG and hydrolyse it to o-nitrophenol.
In part A, IPTG is the inducer and water was used as a control. As seen in the graph, the amount of beta-galactosidase stays the same for 13-15 minutes then increases with time with IPTG as inducer whereas for water it remains almost stagnant. From the graph, the induction time for IPTG is 13-15mins as induction time is when amount of beta-galactosidase is increasing. IPTG is an inducer which will bind to the repressor, causing it to become inactive and hence no blockage of the promoter. (Murray, 2012) With the absence of glucose in sample and IPTG, transcription of the lac operon can proceed hence producing beta-galactosidase.
No change is observed in the water sample as water is not an inducer hence no transcription will occur as repressor is bound to the operator. The water sample hence shows that the lac operon gets transcribe only in presence of an inducer. There is a lag period of the amount of beta-galactosidase produced from the IPTG sample. This is because when IPTG was first added, it takes time for it to go into the cells of E.coli and bind to repressor. Additional time is also needed for transcription to occur. IPTG goes into the cells by the presence of some permease available in cell. (Fernández-Castané, Caminal and López-Santín, 2012)
In part B, samples of different treatment were observed and amount of beta-galactosidase produced for each treatment differs. Allolactose is derived from lactose (Murray,2012) Both IPTG and allolactose are inducers for transcription of beta-galactosidase. Beta-galactosidase is an enzyme that breaks down lactose to galactose and glucose. Hence allolactose is both a substrate and inducer of beta-galactosidase. In graph 2, the amount of beta galactosidase for IPTG and lactose are both increasing which shows that transcription is occurring.
However the amount of beta-galactosidase from IPTG sample is almost twice as much as from the lactose sample. This is due to beta-galactosidase being produced and at the same time breaking down lactose present, hence amount of lactose decreases, resulting in lesser transcription of beta-galactosidase. IPTG is not a substrate for beta-galactosidase and so it is not being hydrolysed by it. The induction of IPTG is more efficient as compared to lactose as it is constantly producing beta-galactosidase and not being hydrolysed.
Both glucose and lactose are a carbon source. For the cell, glucose is the preferred substrate to metabolise as it uses less energy to break it down. However, to metabolise lactose, more energy is needed to produce enzymes to break down lactose. (Deutscher, 2008) In presence of glucose and IPTG, the amount of beta-galactosidase present is much less that those compared to the IPTG sample or the lactose sample as shown in graph 2. This is because glucose is being metabolized instead, there is no need of beta-galactosidase hence less is available. When glucose is present, there is a low cAMP level, and hence no cAMP.CAP complex formed. Without any cAMP.CAP complex binding near to the promoter, no RNA polymerase is stimulated to bind to promoter for transcription to occur. (Murray, 2012)
Varying concentration of IPTG in this case does not affect anything, as in presence of glucose no transcription would occur. From the graph, it can be seen that during the first 10minutes of the experiment, no transcription was occurring, this is because of the absence of the cAMP.CAP complex stimulating transcription. After 10 minutes, amount of beta-galactosidase starts to increase, this is due to glucose being metabolized by cell, hence cAMP levels is high and binds to CAP forming a cAMP.CAP complex that stimulates transcription. This is the positive control, catabolite repression. Also with the addition of IPTG(inducer) transcription is able to take place. At this point, concentration of IPTG would affect the amount of beta-galactosidase present, but from the graph, both samples of IPTG5mM+glucose and IPTG10mM+glucose is the same. This could be an experimental error.
3 samples were treated with IPTG and antibiotics, chloramphenicol, rifampicin and streptomycin. From the graph, in presence of antibiotics, the amount of beta-galactosidase is much less than compared to sample with just IPTG. This shows that these antibiotics inhibit production of beta-galactosidase. All 3 antibiotics have different ways of inhibition. . Chloramphenicol and streptomycin inhibits at the translation phase, rifamicin inhibits at transcription phase. (Neu et al, 1996)
All these antibiotics inhibits protein(beta galactosidase) synthesis. Therefore amount of beta-galactosidase present as compared to just the IPTG sample is much less. Chloramphenicol inhibits ribosomal function. Peptidyletransferase binds onto the 50S ribosome and inhibits peptide bond formation. Streptomycin also inhibits ribosomal function. It cause ribosome to misread the genetic code by binding of a specific protein onto the 30S subunit. Rifampicin inhibits RNA synthesis. The initiation process is disrupted by the binding of rifampicin to subunit of RNA polymerase, hence no RNA synthesised. (Neu et al, 1996) in presence of antibiotic, protein synthesis is inhibited, this shows that the lac operon codes for proteins.
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