7. ColE1 plasmids replicate in enteric bacteria but cannot replicate in Halobacterium salinarium. The bop gene from H. salinarium was cloned into a ColE1 plasmid, and an insertion mutation constructed that disrupted the plasmid encoded bop gene (indicated by bop’-'bop in the figure below). This plasmid was then transformed into H. salinarium with selection for resistance to the antibiotic mevinolin (MevR).
The plasmid does not have any functional replication origin and, except for the bop gene, the plasmid lacks any homology with the H. salinarium chromosome. How do the MevR transformants arise? [Show a diagram and briefly describe your answer.]
ANSWER: The plasmid cannot replicate, so the MevR colonies must arise by integration of the plasmid into the chromosome by homologous recombination between the bop genes.
When the resulting transformants were subsequently grown for many generations without mevinolin (Mev), some Mevs colonies were obtained. About half of these Mevs colonies were Bop+ and half were Bop-. How do the Mevs Bop- colonies arise? [Show a diagram and briefly describe your answer.]
ANSWER: Segregration of the chromosomal bop duplication will yield Mevs colonies. Sometimes the recombination will occur on the same side of the bop disruption as the initial integration event and these will result in bop+ colonies [crossover (i) in the figure below]. Sometimes the recombination will occur on the other side of the bop disruption relative to the initial integration event and these will result in bop- colonies [crossover (ii) in the figure below].
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