1)The role of a reporter gene is very important. This gene has activity that is
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Question
1)The role of a reporter gene is very important. This gene has activity that is easy to detect. It is commonly used to study promoter activity under a variety of investigative circumstances. Recombinant DNA technology is used to attach such a reporter gene to the promoter of interest, after which the recombinant molecule is inserted by transformation or transfection into the cell or host organism of choice. Green Fluorescent protein (GFP) from the jelly fish Aequorea victoria fluoresces green when exposed to blue light. Assume you wish to study the regulatory apparatus of the lactose operon and you develop a plasmid (p) that contained all the elements of the lactose operon except you replace the structural gene with the GFP gene (a reporter gene). You use the plasmid to transform E. coli. Considering the following genotypes, under which the following conditions would you expect Beta-galatosidase production and/or green colonies when examined under blue light. Lactose or Glucose may be present (+) or absent (-) in the media. Please indicate if there is activity of Beta galactosidase and GFP.
Medium condition Respo
Genotype
Lactose
Glucose
Beta galactosidase
Green colonies
I+O+Z+/(p) I+O+ GFP
-
+
-
-
I+O+Z+/(p) I+O+ GFP
+
+
I+O+Z+/(p) I+O+ GFP
+
-
I+O+Z+/(p) ISOC GFP
-
+
I+OCZ+/(p) ISO+ GFP
+
-
I+O+Z-/(p) I+OC GFP
-
+
I+O+Z-/(p) I+OC GFP
+
-
I+O+Z+/(p) ISOC GFP
+
-
I+OCZ+/(p) ISO+ GFP
-
+
Genotype
Lactose
Glucose
Beta galactosidase
Green colonies
I+O+Z+/(p) I+O+ GFP
-
+
-
-
I+O+Z+/(p) I+O+ GFP
+
+
I+O+Z+/(p) I+O+ GFP
+
-
I+O+Z+/(p) ISOC GFP
-
+
I+OCZ+/(p) ISO+ GFP
+
-
I+O+Z-/(p) I+OC GFP
-
+
I+O+Z-/(p) I+OC GFP
+
-
I+O+Z+/(p) ISOC GFP
+
-
I+OCZ+/(p) ISO+ GFP
-
+
Explanation / Answer
if one can understand a few things it is easier to solve these kind of questions.
first comes to I which codes for repressor. there are 3 types - I+ Wild type and is normal and make Z and Y products in the presence of lactose only. I- is a recessive mutation will make products of Z and Y in the presence and the absence of lactose. Is is a dominant mutation will stop both the products Z and Y. if you have atlease one Is in the question neither the products will from no matter whether the lactose is presence or not.
then comes to Operator (O) ,O+ is a wild type and can produce both the products of Z and Y. in the presence of lactose.and Oc is the mutation and can produce both Z and Y products in the Presence and even in the absence of lactose. Oc - constitutive operator to which Repressor cannot bound. it makes Z and Y products even in the absense of lactose. and the mutation Oc is dominant over wild type O+. but constitutive operator allele are Dominant if the other cis-acting elements of the operon are functional means the promoter and coding regions need to be functional.
next comes to Z+ represents production of -galactosidase and Z- represents no production of -galactosidase.
In the above GFP Gene introduced in the place of structural genes but under the regulation of same Inducer and Operator. so the mutants those are discussed above also have the same effect on the expression of GFP
Genotype
Lactose
Glucose
Beta galactosidase
Green colonies
I+O+Z+/(p) I+O+ GFP
-
+
-
-
I+O+Z+/(p) I+O+ GFP
+
+
-
-
I+O+Z+/(p) I+O+ GFP
+
-
+
+
I+O+Z+/(p) ISOC GFP
-
+
-
-
I+OCZ+/(p) ISO+ GFP
+
-
+
-
I+O+Z-/(p) I+OC GFP
-
+
-
+
I+O+Z-/(p) I+OC GFP
+
-
-
+
I+O+Z+/(p) ISOC GFP
+
-
+
-
I+OCZ+/(p) ISO+ GFP
-
+
+
Genotype
Lactose
Glucose
Beta galactosidase
Green colonies
I+O+Z+/(p) I+O+ GFP
-
+
-
-
I+O+Z+/(p) I+O+ GFP
+
+
-
-
I+O+Z+/(p) I+O+ GFP
+
-
+
+
I+O+Z+/(p) ISOC GFP
-
+
-
-
I+OCZ+/(p) ISO+ GFP
+
-
+
-
I+O+Z-/(p) I+OC GFP
-
+
-
+
I+O+Z-/(p) I+OC GFP
+
-
-
+
I+O+Z+/(p) ISOC GFP
+
-
+
-
I+OCZ+/(p) ISO+ GFP
-
+
+
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