Question

1, (12 pts.) You are fortunate to obtain a strain of E. coli from the Pasteur Institute labeled #348, preserved from the work of Jacob & Monod. The genotype of the strain is Lac LacP Laco' Lacz' LacY. The Lacf mutation results in resistance to allolactose allosteric inhibition so it is always bound to the LacO operator. The LacY is a mutation resulting in zero permease activity. Lucky for you, today's echnology allows you to test this bacterial strain to confirm the genotype with two newly developed chemicals. Chemical 1 is Smase which is an allolactose analogue capable of inhibiting Lach mutant protein from binding the LacO operator. Chemical 2 is Ymase and opens large membrane channels in the bacteria which allows for rapid lactose uptake. Fill in the table below for both B-Gal. & Permease activity and determine if the bacteria can grow. Table 1: Predicted effects of treating E. coli strain #34B with Chemicals 1 & 2. Treatment (Lactose B-Gal Activity Permease Activity Can the bacteria grow under these conditions? only) plus: Control (nothing) Smase Ymase Smase +Ymase + or- + or- Explain why or why not (e.g. yes, because...)

Answer 1

ANSWER:

= Note: 1. Here B- gal refers to the enzyme Beta glycosidase that hydrolysis glycoside bond in carbohydrates(Here Lactose) into monosaccharides. Here Lactose----(B gal)------> Glucose+Galactose

2. Allolactose refers to a carbohydrate similar to disaccharide lactose but present with Beta 1,6 linkageinstead of 1,4 linkage seen in lactose

Treatment	B-Gal activity + or -	Permease activity + or -	Explanation
Control	+	-	NO. Grows until all the lactose inside the cell is depleted. For Explanation, Please refer to point 1 mentioned below the table
Smase	-	-	NO. For Explanation, Please refer to point 2 mentioned below the table
Ymase	+	+	YES. For Explanation, Please refer to point 3 mentioned below the table
Smase+Ymase	-	+	NO. For Explanation, Please refer to point 4 mentioned below the table

1. In this case, the bacteria is not treated with anything. The Given that the genotype is Y- is a mutation which results in zero permease activity which means that ​Lactose cannot be taken up by the cell, The Bacteria will grow only for a certain period of time until all the lactose inside of the cell is depleted. Hence the answer is BACTERIA GROWS BUT ONLY FOR A LIMITED PERIOD OF TIME. B-gal is answered as +ve since it can hydrolyse the endocytic lactose, but soon after the substrate concentration decreases due to the zero permease(Lac Y- mutation).

2. in the second case which is treated with Smase, which is a allolactose analogue as explained earlier it inhibits the LaCL^S​to bind to LacO operator. Hence the enzyme B-gal is allosterically inhibited(Hence answer in table is -ve) resulting in its decreased activity. Since LacY- is not changed in this case, even the permeation of lactose is also -ve. The outcome of this condition is BACTERIA CANNOT GROW. Hence answer is NO

3. In the 3rd case, the treatment with Ymase results in more uptake of lactose, due to high permease activity(Hence +) and since LaCLS​ is binded to LacO the enzyme B-gal efficiently binds to the substrate lactose(Hence +ve or B-gal) . The outcome is Bacteria thrives(Grows) in the medium treated with Ymase

4. When the medium is treated with both Smase and Ymase simultaneously, permease activity(+ve) is enhanced by Ymase, but Smase offers susceptibility of allolactose allosteric inhibition resulting in decreased B-gal activity (-ve). Hence the answer is NO, population does not grow due to the hyper uptake of lactose into the cell which may lead to Cytolysis,, since lactose is not being hydrolysed by B-gal