The following table lists 4 bacterial strains that are partial diploids for lac operon genes. Given the activity of beta-galactosidase measured for each strain in the absence (-lac) or presence (+lac) of lactose, complete the table by choosing the appropriate symbol (+, -, C, S) to indicate the allele of the gene or site missing from the table (blue numbers). + = wildtype, - = null mutation, c = constitutive, s =super repressor chromosome plasmid B-gal act. strain 10 Z A 1 C B 3 4 + C + 6 D 9 + 1 [Select] 3 [Select] 5 [Select] 7 [Select] 9 [Select] I 0 2 + + 5 + - Z -lac + 0.062 + 0.003 7 8 0.062 10 + 0.003 2 [Select] 4 [Select] 6 [Select] 8 [Select] 10 [Select] +lac 0.058 0.004 0.117 0.060

Based on the partial diploid sketched shown in Figure 3, where cross-hatched rectangles and the red bar indicate mutations that inactivate that particular genetic element of the operon, state if expression of LacO (Lac Operator) and LacY (Lac Permease) is (select one of the options below for each genetic element and briefly justify your answer): • constitutively ON (in the presence/absence of lactose) • constitutively OFF (in the presence/absence of lactose) • inducible by lactose • not enough information given to conclude • none of the above FIGURE 3: lact laco lacz- lacř lact laco lacz lac

Give the levels of Beta-galactosidase activity (high or low or zero) expected for the following partial diploids for the lac operon strains.

Strain ROFL4 has a premature stop mutation in the lacZ gene, resulting in a nonfunctional b-galactosidase. Otherwise all other parts of the operon are functional. Circle the least number of components for an F' plasmid that will restore normal regulation and function of the lac operon in the resulting partial diploid. (may need more than one) (a) lacI+   (b) lacO+   (c) lacP+   (d) lacZ+   (e) None, cannot be restored.

Examine the following theoretical operon (gen) in E. coli which contains structural genes (genC, genB, genA) that are involved in amino acid biosynthesis. The gen operon also includes the gene encoding for the regulator molecule within it. When the amino acid that this operon helps synthesize is present, it binds to the regulator forming a complex which can bind to the operator region. Assume that the regulator molecule only binds to the operator in the presence of the amino acid. Promoter || Operator genC genB gen operon Regulator genA

For the following genotypes of E. coli strains, state whetherenzyme activity is inducible (I), constitutive (C), or uninducible (U).Key to symbols in table:z = structural gene for  -galactosidasey = structural gene for permeasea = structural gene for transacetylasep = promoter (for lac operon)i = structural gene for the lac repressor (regulator gene)o = operator (binding site for repressor)Genotype                                 -gal (z)                       Permease (y)                         Transacetylase (a)i+ p+ oC z- y- a+i- p+ o+ z+ y- a- /i+ p- oc z- y+ a-i- p+ o+ z+ y- a+ /i+ p+ oc z- y+ a-i+ p+ o+ z- y- a+i- p- o+ z+ y- a+ /i- p+ o+ z- y+ a-iS p+ o+ z- y- a+i- p+ o+ z+ y- a+ /i+ p- oc z- y+ a-is p+ o+ z- y+ a+ /i+ p+ oc z+ y- a-i- p- o+ z+ y- a+ /i- p+ o+ z- y- a-is p+ oc z+ y- a+i+ p+ oc z+ y- a+

O The lac operon in E.coli encodes enzymes necessary for the breakdown of lactose. For each enzyme (lac Z and lac Y), indicate with a + or-whether or not it is made when there is no lactose or when there is lactose. B-galactosidase (lac Z) No Lactose Permease (lac Y) Lactose Lactose No Lactose Genotype PP0 Z Y/I P*O*Z•Y* I'POCZ Y*/I P* O©Z*Y° P O Z'Y/I P'OʻZ'Y* PP O ZY*/IP*O*Z*Y* IP OCZ Y /I P*O*Z•Y* IPO ZY*/I* P*O©Z*Y• I'PO*Z Y*/IP'O*Z*Y°

To characterize the promoter of the gadA operon you made a series of deletion mutants removing pieces of the promoter to see what would happen. The results are found below: gad promoter gada gadX gadz 450 +1 lacz activity transcription start site pH 2.0 pH 7.0 A gad promoter beta-galactosidase (lacZ) +++ 450 gad promoter beta galactosidase (lacZ) +++ +++ 300 +1 gad promoter beta galactosidase (lacZ) 150 D gad beta galactosidase (lacz) -450 150 E gad promoter beta-galactosidase (lacZ) -450 -300 Based on these results, what can you conclude about the gad promoter? O a. The promoter is only regulated by repression Ob. The promoter is regulated by a mix of activation and repression O c. The promoter is only regulated by activation O d. The promoter has multiple operators and multiple enhancers

Consider this strain of E. coli with lac operon alleles: IS pt o* z* y+ /T p* oCz- Y+ In a written response, briefly explain if beta-galactosidase AND permease will be expressed, and if so, will they be inducible or constitutive.

A mutant E. coli strain is isolated which does not ever produce the enzymes β-galactosidase or permease, irrespective of whether lactose is present or absent. If the normal wild-type genotype with respect to the lac operon can be represented as lacI+ lacP+ lacO+ lacZ+ lacY+, give three different mutant genotypes (list all 5 operon components in each case, do not consider partial diploids) which would result in the mutant phenotype described here.