MCB 2050 Midterm Questions and Answers
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4) Describe what happens to a colony containing a plasmid with the insert. Correct Answ_1)
insertional inactivation
2) it encodes beta-galactosidase which convert the colourless substrate Xgal into a blue product
3)- functional lacZ gene
-functional beta-galactosidase enzymes convert X-gal (colourless) to a blue coloured product
-colonies are blue
4) -non functional lacZ gene
-functional beta-galactosidase enzyme not made
-colonies are white
1) What are expression plasmids designed for?
2) What must they be able to do?
3) What must they contain (3)? Correct Answ_1) the expression of proteins in bacteria, yeast,
plant, or animals cells
2) replicate in bacteria in order to amplify the plasmid
3) -a selectable marker to identify bacteria containing the plasmid
,-a cloning site located downstream from a promoter appropriate for the host cell to allow the
inserted gene to be expressed
-a selectable marker (antibiotic resistance gene) appropriate for the host cell to allow for
selection of cells that have incorporated the plasmid in their genome
1) Do promoters have directionality?
2) What must eukaryotic expression plasmids contain? Correct Answ_1) Yes
2) a promoter upstream of the MCS and a polyA signal downstream of the MCS
1) What does MCS allow for?
2) What do reporter genes code for?
3) What are reporter genes used for?
4) What does luc+=?
5) What is done to the luciferase protein? (2)
6) What is the luciferase protein proportional to?
7) Does the reporter gene have a promoter to control its expression?
8) What happens at the MCS?
9) What is the reporter gene called in this example? Correct Answ_1) the insertion of
potential gene regulatory sequences
,2) a protein that can be visualized or measured
3) to study gene regulatory sequences after transfection into cells
4) the firefly luciferase gene
5) -it is assayed (converts luciferin to light)
-that light can be measured in a luminometer
6) [light] proportional to [luciferase protein] proportional [luciferase mRNA] which is
proportional to transcriptional activity
7) No
8) insert gene regulatory sequences here
9) firefly luciferase (luc)
Compare the reporter plasmid and expression plasmid (2 for expression, three for reporter)
Correct Answ_Reporter Plasmid:
-the reporter gene does not have a promoter to control its expression
-MCS: insert regulatory gene sequences here
-the expression level of the reporter gene will tell you the efficiency of the regulatory sequences
that you inserted
Expression Plasmid:
-promoter to control expression of your gene of interest
, -MCS: insert gene of interest (the gene you want to express) here
1) Describe cloning plasmids and give an example.
2) Describe expression plasmids and give an example.
3) Describe reporter plasmids and give an example. Correct Answ_1) -for isolation and
amplification of DNA sequences
-contains (i) origin of replication, (ii) selectable marker and (iii) MCS
-example: pBluescript (also has blue/white selection for plasmids without/with inserts)
2) -for the expression of proteins in bacteria, yeast, plant or animal cells
-contains promoter appropriate for target cell located upstream of MCS
-also contains elements found in cloning vector (for cloning, amplification of E.coli)
-example: pCMV, a mammalian cell expression vector
3) -for the measurement of gene regulatory sequences
-contains a reporter gene that when expressed can be quantitatively measured
-MCS is located upstream of the reporter gene
-also contains elements found in cloning vector
-example: pGL3 (contains the luciferase reporter gene)
100% Solved
4) Describe what happens to a colony containing a plasmid with the insert. Correct Answ_1)
insertional inactivation
2) it encodes beta-galactosidase which convert the colourless substrate Xgal into a blue product
3)- functional lacZ gene
-functional beta-galactosidase enzymes convert X-gal (colourless) to a blue coloured product
-colonies are blue
4) -non functional lacZ gene
-functional beta-galactosidase enzyme not made
-colonies are white
1) What are expression plasmids designed for?
2) What must they be able to do?
3) What must they contain (3)? Correct Answ_1) the expression of proteins in bacteria, yeast,
plant, or animals cells
2) replicate in bacteria in order to amplify the plasmid
3) -a selectable marker to identify bacteria containing the plasmid
,-a cloning site located downstream from a promoter appropriate for the host cell to allow the
inserted gene to be expressed
-a selectable marker (antibiotic resistance gene) appropriate for the host cell to allow for
selection of cells that have incorporated the plasmid in their genome
1) Do promoters have directionality?
2) What must eukaryotic expression plasmids contain? Correct Answ_1) Yes
2) a promoter upstream of the MCS and a polyA signal downstream of the MCS
1) What does MCS allow for?
2) What do reporter genes code for?
3) What are reporter genes used for?
4) What does luc+=?
5) What is done to the luciferase protein? (2)
6) What is the luciferase protein proportional to?
7) Does the reporter gene have a promoter to control its expression?
8) What happens at the MCS?
9) What is the reporter gene called in this example? Correct Answ_1) the insertion of
potential gene regulatory sequences
,2) a protein that can be visualized or measured
3) to study gene regulatory sequences after transfection into cells
4) the firefly luciferase gene
5) -it is assayed (converts luciferin to light)
-that light can be measured in a luminometer
6) [light] proportional to [luciferase protein] proportional [luciferase mRNA] which is
proportional to transcriptional activity
7) No
8) insert gene regulatory sequences here
9) firefly luciferase (luc)
Compare the reporter plasmid and expression plasmid (2 for expression, three for reporter)
Correct Answ_Reporter Plasmid:
-the reporter gene does not have a promoter to control its expression
-MCS: insert regulatory gene sequences here
-the expression level of the reporter gene will tell you the efficiency of the regulatory sequences
that you inserted
Expression Plasmid:
-promoter to control expression of your gene of interest
, -MCS: insert gene of interest (the gene you want to express) here
1) Describe cloning plasmids and give an example.
2) Describe expression plasmids and give an example.
3) Describe reporter plasmids and give an example. Correct Answ_1) -for isolation and
amplification of DNA sequences
-contains (i) origin of replication, (ii) selectable marker and (iii) MCS
-example: pBluescript (also has blue/white selection for plasmids without/with inserts)
2) -for the expression of proteins in bacteria, yeast, plant or animal cells
-contains promoter appropriate for target cell located upstream of MCS
-also contains elements found in cloning vector (for cloning, amplification of E.coli)
-example: pCMV, a mammalian cell expression vector
3) -for the measurement of gene regulatory sequences
-contains a reporter gene that when expressed can be quantitatively measured
-MCS is located upstream of the reporter gene
-also contains elements found in cloning vector
-example: pGL3 (contains the luciferase reporter gene)
