Β-Galactosidase Lab Report
Gene Expression
Introduction:
Escherichia coli are capable of using lactose as their sole carbon source. E. coli produces the enzyme β-galactosidase to digest the lactose into glucose and galactose. However, it would be inefficient to produce enzymes when there is no lactose available, or if there is a more readily-available energy source available such as glucose. Therefore there must be something controlling the expression of this enzyme. The purpose of the experiment is to determine whether induction of β-galactosidase is due to new enzyme being made or due to activation of pre-existing enzyme. To determine if a new enzyme was being made we used the compounds 5-fluorouracil (5-FU) and chloramphenicol (Cm) as transcription and translation …show more content…
inhibitors. A non-metabolisable analogue of lactose, isopropyl-β-D-thiogalactoside (IPTG), was also added to one of the samples to see if the bacteria showed a similar response.
Results:
Figure 1.
Specific activity of enzyme β-galactosidase. E. coli cells were incubated at 37°C until it had at least an A600nm of 0.08 in a phosphate buffer, pH 7.0 containing ammonium sulphate, sodium citrate, MgSO4, thiamine and glycerol. Six treatments were applied to the E. coli cells 0.5 mM of IPTG, 0.4 mM lactose, 0.4 mM lactose and 5.6 mM glucose, 0.4 mM lactose and 1 mg/mL 5-FU, 0.4 mM lactose and 1mg/mL CM and the last treatment was a control. Protein concentration was estimated over a 35 minute time period using the equation A600*150/1.4. During the same time period samples extracted from the treatments are mixed immediately with 50 mM sodium phosphate buffer, pH 7.0; 5 mM KCl; 1mM MgSO4; 50 mM 2-mercaptoethanol, 0.05% (v/v) chloroform and 0.05% (w/v) SDS, vortex for 10 seconds. This is to stop any further induction of the β-galactosidase. Pre-incubate at 28°C to ensure the assay tubes are equilibrated for the assay incubation. Β-galactosidase activity was estimated in lysed cells by measuring the rate of appearance of o-nitrophenolate (oNP) in an assay containing 2.7 mM o-nitrophenol galactose (oNPG) as substrate. The reaction was stopped by the addition of 0.4 M Na2CO3 and vortexed. The absorbance of the tubes were taken at 420nm and 550nm (ε=4.5 mM-1 cm-1) using the latter reading to correct for
turbidity.
Conclusion
The results suggest that that the induction of β-galactosidase is due to new enzymes being made and not due to pre-existing enzymes. This is shown by reduced levels of β-galactosidase activity in the presence of both chloramphenicol and 5-FU which are translation and transcription inhibitors. This suggests that it needs these mechanisms to produce β-galactosidase and therefore is not a pre-existing enzyme. The activity in the IPTG treatment shows that β-galactosidase was expressed twice as much as the lactose treatment this may be because it is non-metabolisable so has an increased amount of substrate. This might be stimulating the production of β-galactosidase. The reduced activity of the lactose verse the lactose and glucose treatment by nearly 3 fold suggests that the glucose is the preferred carbon source for E. coli. Glucose would be favoured in this situation because it requires less processing to be used as an energy source. Whereas lactose first has to be broken down by β-galactosidase to produce glucose and galactose. By having an alternative food source the β-galactosidase is only partially expressed. The significantly low levels of activity in the inhibitor treatment strongly suggest that β-galactosidase is the result of new molecules being made through translation and transcription.
Introduction:
Escherichia coli are capable of using lactose as their sole carbon source. E. coli produces the enzyme β-galactosidase to digest the lactose into glucose and galactose. However, it would be inefficient to produce enzymes when there is no lactose available, or if there is a more readily-available energy source available such as glucose. Therefore there must be something controlling the expression of this enzyme. The purpose of the experiment is to determine whether induction of β-galactosidase is due to new enzyme being made or due to activation of pre-existing enzyme. To determine if a new enzyme was being made we used the compounds 5-fluorouracil (5-FU) and chloramphenicol (Cm) as transcription and translation …show more content…
inhibitors. A non-metabolisable analogue of lactose, isopropyl-β-D-thiogalactoside (IPTG), was also added to one of the samples to see if the bacteria showed a similar response.
Results:
Figure 1.
Specific activity of enzyme β-galactosidase. E. coli cells were incubated at 37°C until it had at least an A600nm of 0.08 in a phosphate buffer, pH 7.0 containing ammonium sulphate, sodium citrate, MgSO4, thiamine and glycerol. Six treatments were applied to the E. coli cells 0.5 mM of IPTG, 0.4 mM lactose, 0.4 mM lactose and 5.6 mM glucose, 0.4 mM lactose and 1 mg/mL 5-FU, 0.4 mM lactose and 1mg/mL CM and the last treatment was a control. Protein concentration was estimated over a 35 minute time period using the equation A600*150/1.4. During the same time period samples extracted from the treatments are mixed immediately with 50 mM sodium phosphate buffer, pH 7.0; 5 mM KCl; 1mM MgSO4; 50 mM 2-mercaptoethanol, 0.05% (v/v) chloroform and 0.05% (w/v) SDS, vortex for 10 seconds. This is to stop any further induction of the β-galactosidase. Pre-incubate at 28°C to ensure the assay tubes are equilibrated for the assay incubation. Β-galactosidase activity was estimated in lysed cells by measuring the rate of appearance of o-nitrophenolate (oNP) in an assay containing 2.7 mM o-nitrophenol galactose (oNPG) as substrate. The reaction was stopped by the addition of 0.4 M Na2CO3 and vortexed. The absorbance of the tubes were taken at 420nm and 550nm (ε=4.5 mM-1 cm-1) using the latter reading to correct for
turbidity.
Conclusion
The results suggest that that the induction of β-galactosidase is due to new enzymes being made and not due to pre-existing enzymes. This is shown by reduced levels of β-galactosidase activity in the presence of both chloramphenicol and 5-FU which are translation and transcription inhibitors. This suggests that it needs these mechanisms to produce β-galactosidase and therefore is not a pre-existing enzyme. The activity in the IPTG treatment shows that β-galactosidase was expressed twice as much as the lactose treatment this may be because it is non-metabolisable so has an increased amount of substrate. This might be stimulating the production of β-galactosidase. The reduced activity of the lactose verse the lactose and glucose treatment by nearly 3 fold suggests that the glucose is the preferred carbon source for E. coli. Glucose would be favoured in this situation because it requires less processing to be used as an energy source. Whereas lactose first has to be broken down by β-galactosidase to produce glucose and galactose. By having an alternative food source the β-galactosidase is only partially expressed. The significantly low levels of activity in the inhibitor treatment strongly suggest that β-galactosidase is the result of new molecules being made through translation and transcription.