Concept explainers
A plasmid that is both ampicillin and tetracycline resistant is cleaved with Pstl, which cleaves within the ampicillin resistance gene. The cut plasmid is ligated with Pstl-digested Drosophila DNA to prepare a genomic library, and the mixture is used to transform E. coli K12.
- (a) Which antibiotic should be added to the medium to select cells that have incorporated a plasmid?
- (b) If recombinant cells were plated on medium containing ampicillin or tetracycline and medium with both antibiotics, on which plates would you expect to see growth of bacteria containing plasmids with Drosophila DNA inserts?
- (c) How can you explain the presence of colonies that are resistant to both antibiotics?
(a)
To determine: The antibiotic that an individual should add to the medium to select cells that contain the recombinant plasmid.
Introduction: Plasmid refers to an circular, extrachromosomal DNA (deoxyribonucleic acid) molecule that replicates chromosome independently.
Explanation of Solution
The gene that confers resistance to tetracycline is intact in the recombinant plasmid. An individual would add tetracycline to the medium to select the recombinant plasmd in the cells. In the medium, the bacteria that have been transformed with recombinant plasmid will be resistant to tetracycline.
Thus, an individual would use tetracycline to select cells that contain the recombinant plasmid.
(b)
To determine: The plates on which an individual might see the growth of bacteria having plasmids with Drosophila DNA inserts.
Introduction: Genetically modified bacterial plasmids were the first developed vectors, used for cloning purpose.
Explanation of Solution
Cloning is a important process in which bacterial cells containing recombinant DNA can be readily identified. This process is also accomplished is through the use of selectable marker genes. Antibiotic resistant genes provide very effective selectable marker genes. Therefore, the colonies that grow on tetracycline medium but do not grow on ampicillin medium would contain the Drosophila DNA insert.
Thus, colonies that only grow in tetracycline medium probably contain the Drosophila DNA insert.
(c)
To determine: The reason that some colonies can grow on tetracycline medium as well as ampicillin medium.
Introduction: Plasmids have an origin of replication (ori) site, which makes it possible to produce several hundred copies of a plasmid in a single host cell.
Explanation of Solution
When performing cloning process, it is not necessary for incorporation of all DNA plasmids need to be cloned. A plasmid cut with a restriction enzyme generating sticky ends can self-ligate if cut ends of the plasmid rejoin. Also, if cleavage with PstI was incomplete, then no change in biological characteristics of the uncut plasmid would be expected.
Thus, self-ligation of plasmid and incomplete cleavage with PstI are responsible for the growth of colonies that are resistant to both antibiotics.
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Chapter 20 Solutions
Concepts of Genetics (12th Edition)
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