Gene Expression

Gene expression II

Introduction:

IPTG (Isopropyl ?-D-1-thiogalactopyranosid) is a compound that is used as an inducer of lac operon. It can enter the cell without the help of membrane proteins. IPTG induces production of ?-galactosidase by binding and inhibiting the lac repressor. IPTG is not a substrate of ?-galctosidase, thus, its concentration remains constant. The aim of this experiment was to examine the mechanism of IPTG induction and compare it with lactose induction the hypothesis for this experiment is that both mechanisms are the same.

Results:

Figure1: The effect of different compounds on the specific activity of ?-galactosidase iduced by IPTG over a period of time.

Number of compounds was used to treat E-coli cultures grown in glycerol medium. Cultures were incubated for 32 min. the compounds were 0.5 mM isopropyl-?-D-thiogalactoside (IPTG), 0.4 mM lactose, 0.5 mM (IPTG) and 5 mM glucose, 0.5 mM (IPTG) and a5-fluorouracil (5-FU) (24 ug/mL), 0.5 mM (IPTG) and chloramphenicol (CM) (24 ug/mL) and a control with no addition.

During the incubation time, protein concentration and ?-galactosidase activity were measured. The protein concentration was recorded at an absorbance of 600 nm and calculated using the conversion factor: A260 *150/1.4. ?-galactosidase activity in each culture was evaluated by measuring the rate of formation of o-nitrophenolate (o-NP) at 28 °C in an assay containing 2.2 mM of the substrate o-nitrophenolate galactose (o-NPG). Assays were performed in 50 mM sodium phosphate, pH 7.0; 5mM KCl; 0.5 mM MgSO4 and 25 mM 2-mercaptoethanol. Then, by adding Na2CO3 (294mM) as a colour gradient was achieved the reaction was stopped. ONP formation was measured at 420 nm and corrected for turbidity at 550 nm as well as using the extinction coefficient of 4.5 mM-1cm-1. (Correction factor: A420- (1.3*A550)).

Discussion:

Figure 1 shows that the specific activity of ?-galactosidase induced by lactose and IPTG in the absence of inhibitors and glucose is caused by newly synthesised enzymes. In addition, the specific activity in the culture treated with IPTG only is higher than the culture treated with lactose. This is due to the rate of transporting