Induction of Gene Expression - Research Paper Example

They are preceded by a single promoter/operator region which controls the expression of all three genes. Even further upstream of the promoter lies the gene lacI which codes for the lac repressor protein. This protein is a regulator and binds to the promoter/operator region of the lac operon in the absence of lactose in the medium. This is simply an economic measure by the bacterium to prevent the wasteful synthesis of enzymes when they are not needed. In the presence of lactose, the repression is relieved as lactose binds to the repressor protein and changes its conformation in a manner that makes it dissociate from the promoter.

However, there is an added control level to the regulation of this operon. In the presence of a preferred substrate, like glucose or its modified form, glucose -6- phosphate, the bacterium will still not synthesize lactose even though this is present in the medium along with glucose. This phenomenon is called catabolite repression. The mechanism involves the CAP protein which also can increase expression of the lac operon. When glucose levels are high, cyclic AMP levels lower. Cyclic AMP forms a complex with CAP before it binds to the DNA.

So, when the cyclic AMP levels are lowered, the CAP protein bound to DNA also decreases, thus lowering the transcription of lac genes. . Since the natural substrate lactose and the products of its metabolism are not coloured detection of their formation is difficult. For this purpose, the analog ONPG is used which upon hydrolysis yields a product which is deep yellow in colour and can be spectrophotometrically quantified to follow the reaction and hence the expression patterns of the operon. The aim of the work is to use this analog and others to obtain a better understanding of the workings of the lac operon.

MATERIALS AND METHODS Culturing of the bacteria: 10 ml each of E. coli (lac+ strain) which had reached mid-log phase was aliquoted into separate flasks and incubated at 28- 30 C gently shaking to ensure aeration. The cells were allowed to continue growing.Induction:Two sets of induction experiments were performed. The first set was induced with IPTG at a final concentration of 0.5 mM. The second set was also induced with 0.5 mM IPTG but in addition glucose was added to the medium to a final concentration of 30 mM.

For the first set 1 ml samples were taken out at intervals of 2, 4, 8, 16, 24, 32 and 48 minutes and used for the assay of galactosidase activity. For set 2, samples were removed at intervals of 10 and 45 minutes after induction with IPTG. As a control, 1 ml of the culture was removed prior to induction from both sets and used as uninduced controls.-galactosidase assays:To determine whether expression of the operon was taking place, the activity of galactosidase was assayed as follows. 0.1 ml of the culture samples removed at each time point were transferred into spectrophotometer tubes appropriately labeled. 1.5 ml of ONPG assay medium was added to each tube.

(100 ml of assay medium contains 8 mg ONPG, 0.1 ml mercaptoethanol, 0.001 M MgSO4, pH7). After brief vortexing