Gene Expression

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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 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...
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