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An operon is a group of genes that are arranged side by side with a regulatory gene.
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It also includes a promoter gene, operator gene, and structural genes. Regulatory genes control transcription with positive or negative signals. This control of gene expression allows organisms to conserve energy by producing only the necessary enzymes.
The lac operon, found in E. Coli, consists of a promoter, operator, regulatory, and structural genes. The structural genes code for enzymes like beta-galactosidase, permease, and thiogalactoside transacetylase, which are involved in lactose metabolism.
The regulatory gene, lacI, produces the repressor that negatively controls gene expression.
The lac operon's transcription occurs when there is no repressor bound to the operator and when the cAMP.CAP complex is bound to the promoter. In the presence of an inducer like IPTG, the repressor and inducer bind together, becoming inactive and allowing transcription to proceed.
This experiment investigates the induction time of the lac operon in E. Coli using IPTG and compares the amount of beta-galactosidase produced with lactose, IPTG, and antibiotics.
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.
The following materials were used in the experiment:
The experimental procedures followed the guidelines outlined in the BIOC2201 Principles of Molecular Biology – Advanced Laboratory Manual (pages 51-61).
There are two parts to this experiment. In part A, the induction time for beta-galactosidase in E. Coli is determined. Water is used as a control. The results, including absorbance readings and corrected absorbance readings, are shown in Table 1, along with the units of beta-galactosidase per ml of bacterial culture.
| Time (mins) | Absorbance Reading | Corrected Absorbance Reading | Beta-galactosidase Units (units/ml) |
|---|---|---|---|
| 0 | 0.200 | 0.195 | 125.64 |
| 5 | 0.220 | 0.215 | 138.35 |
| 10 | 0.245 | 0.240 | 154.52 |
| 15 | 0.285 | 0.280 | 179.94 |
From the data, the induction time for IPTG is approximately 13-15 minutes.
The results for Part B, including the comparison of beta-galactosidase induction with different treatments, are presented in Table 2 below:
| Treatment | Beta-galactosidase Units (units/ml) |
|---|---|
| IPTG | 250.80 |
| Lactose | 128.40 |
| Glucose | 87.60 |
| IPTG + Glucose | 90.20 |
| IPTG + Chloramphenicol | 45.70 |
| IPTG + Rifampicin | 42.80 |
| IPTG + Streptomycin | 48.90 |
In this experiment, the induction time of the lac operon in E. Coli was investigated using IPTG as an inducer. The results showed that the amount of beta-galactosidase remained relatively constant for the first 13-15 minutes and then increased over time when IPTG was used as the inducer. In contrast, the control with water showed little change, indicating that transcription of the lac operon only occurs in the presence of an inducer.
The lag period observed in the IPTG sample is likely due to the time required for IPTG to enter the E. Coli cells and bind to the repressor protein. Additionally, transcription and translation processes take time to produce beta-galactosidase. This delay in induction is a characteristic feature of lac operon regulation.
In part B, the experiment compared the induction of beta-galactosidase by IPTG and lactose. Both IPTG and lactose serve as inducers for the lac operon, but their effects differed. IPTG, unlike lactose, is not a substrate for beta-galactosidase and, therefore, does not get hydrolyzed. This results in more efficient induction with IPTG, as it continuously produces beta-galactosidase without being consumed as a substrate.
When glucose is present, it is the preferred carbon source for the cell, and lactose metabolism requires more energy. This preference is reflected in the lower levels of beta-galactosidase when glucose is available. The catabolite repression mechanism ensures that the lac operon is only transcribed when glucose is scarce and cAMP levels are high.
The experiment also examined the effect of antibiotics, such as chloramphenicol, rifampicin, and streptomycin, on beta-galactosidase production. All three antibiotics significantly reduced beta-galactosidase levels, indicating that they inhibit protein synthesis. Chloramphenicol and streptomycin inhibit ribosome function at the translation phase, while rifampicin disrupts RNA synthesis at the transcription phase.
Overall, this experiment provides valuable insights into the regulation of the lac operon and the factors that influence beta-galactosidase production. It demonstrates the importance of inducers, substrate availability, and catabolite repression in controlling gene expression in E. Coli.
In conclusion, this experiment investigated the induction time of the lac operon in E. Coli using IPTG as an inducer. The results indicated that IPTG effectively induced beta-galactosidase production, with an induction time of approximately 13-15 minutes. Additionally, the experiment compared the induction of beta-galactosidase by IPTG and lactose, showing that IPTG was a more efficient inducer due to its non-substrate nature.
Furthermore, the experiment demonstrated the impact of glucose availability on beta-galactosidase production, highlighting the role of catabolite repression in gene regulation. The inhibitory effects of antibiotics on beta-galactosidase synthesis were also observed, emphasizing the importance of protein synthesis inhibition in antibiotic action.
Based on the findings of this experiment, it is recommended to further investigate the kinetics of lac operon induction with varying concentrations of IPTG and different carbon sources. Additionally, exploring the transcription and translation processes in more detail could provide a deeper understanding of gene regulation in E. Coli.
Further research could also focus on the practical applications of these findings, such as optimizing inducer concentrations for biotechnological processes and understanding how antibiotic resistance mechanisms may be influenced by the regulation of essential genes.
Investigation of Induction Time of lac operon in E. Coli with IPTG. (2016, Nov 27). Retrieved from https://studymoose.com/document/beta-galactosidase-report
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