BETA GLAC - Courtney Docherty Seat 12 Induction...

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Courtney Docherty Seat 12 April 16, 2009 Induction of β -Galactoside Objective: In this experiment we are measuring the activity of the lac-operon by the use β -Galactosidase, which will cleave ONPG. When the ONPG is cleaved it will produce glucose and ONP. We will measure using absorbance. ONP is yellow at sufficiently alkaline pH and ONPG and glucose are clear. So the more ONP present the higher the absorbance will be. Methods: 1. Preparation for the experiment: a. Each test tube will be labeled in five minute increments starting a 0 and going up to 40 b. Each of the tubes will have .9 ml of reducing buffer with sarkosyl. Then each tube will have a drop of toluene added to it and then will remain capped. i. The sarkosyl is added to remove the cell membranes and the toluene is added to remove the cell walls. 2. Sampling the Induced Culture a. .5 ml of 2 mM IPTG solution (experimental group) will be added to the 5 ml of E.coli. Then it will be mixed.
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i. The IPTG is added to our experiment because it is a better inducer then lactose. It is a better inducer because it gets into the cells readily without help from the permease and it is not a substrate for β -Galactoside. ii. If we used lactose as the inducer instead of IPTG there would be along induction lag. Lactose negative strains of E. coli lack galactoside permease activity. The permease activity eliminates most of the lag time before enzyme synthesis begins. So if no permease activity is present then there would be a very long induction lag.
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