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 Plus Mastering Genetics with Pearson eText -- Access Card Package (12th Edition) (What's New in Genetics)
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- A plasmid, pUC18, contains the ampicillin-resistance gene, the origin of replication, and the ß - gal gene, which codes for the B-galactosidase protein. This protein can break down the synthetic chemical X-gal, producing a blue product that stains the entire cell blue (but is harmless to the bacteria). At the beginning of the B-gal gene there are several unique restriction sites (some of them are shown in the diagram below). You wish to clone a 1.0-kb Xbal fragment into the pUC18 plasmid, so you cut the plasmid with Xbal and, after removing the enzyme, mix the Xbal-cut plasmid with the 1.0-kb fragment, ligate, and transform competent bacteria. Pati Xbal EcoRI B-gal A Amp ori Figure: pUC18 plasmid map (a) On what medium would you grow your transformed bacteria? (b) Do you expect the bacteria carrying plasmid pUC18 (without the insert) to be blue or white when grown in the presence of X-gal? Explain.arrow_forwardMany resistance mechanisms are encoded on plasmids. These mechanisms are of great clinical significance, because they can spread very easily through horizontal gene transfer. A culture of the bacterial isolate is grown, and plasmid DNA is isolated using a spin column-based solid phase extraction method. The purified plasmid DNA is then submitted for next-generation sequencing. Bioinformatic analyses of the sequencing results suggests that the following gene is likely involved in antibiotic resistance: > putative antibiotic resistance gene ATGCGTGTATTAGCCTTATCGGCTGTGTTTTTGGTGGCATCGATT ATCGGAATGCCTGCGGTAGCAAAGGAATGGCAAGAAAACAAAAGT TGGAATGCTCACTTTACTGAACATAAATCACAGGGCGTAGTTGTG CTCTGGAATGAGAATAAGCAGCAAGGATTTACCAATAATCTTAAA CGGGCGAACCAAGCATTTTTACCCGCATCTAGTGCGAAAATTCCC AATAGCTTGATCGCCCTCGATTTGGGCGTGGTTAAGGATGAACAC CAAGTCTTTAAGTGGGATGGACAGACGCGCGATATCGCCACTTGG AATCGCGATCATAATCTAATCACCGCGATGAAATATTCAGTTGTG CCTGTTTATCAAGAATTTGCCCGCCAAATTGGCGAGGCACGTATG…arrow_forwardIn this western blot, the levels of phosphorylated TBK (PTBK) decrease with increasing amounts/expression of the viral protein (VP). Figure description: Increasing amounts of a plasmid expressing the viral protein (0.5, 1, or 2ug) were cotransfected with TBK1 expression plasmid. Cells were harvested 24 h post-transfection and analyzed for phosphorylated TBK1 (anti- TBK1 Ser172), total TBK1 (anti-TBK1), B-actin (anti-B-actin), and viral protein (anti-VP) expression by Western blot analysis. VP PTBK1 (S172) TBK1 actin Virus proteins O True O Falsearrow_forward
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