7). (18 points). Shimada, Weisberg, and Gottesman (1973; J. Mol. Biol. 80:297-314) isolated a secondary
site lambda lysogen where the prophage had inserted into the E. coli trpC gene as shown below. The
orientation of the prophage relative to the trp operon as well as some lambda markers in the prophage are
shown. All lambda genes are wild type except for the cI gene which is the temperature sensitive cI857
allele. The lysogen was isolated as a tryptophan auxotroph at 30o C.
A). (6 Points). Why is this strain a tryptophan auxotroph?
The prophage has inactivated the trpC gene which is required for tryptophan biosynthesis.
If a culture of this lysogen is grown at 30o in LB broth and an aliquot is plated on LB plates which
are incubated at 42o C, rare colonies occur at a frequency of about 1 in 107 cells plated. Colonies
are picked and checked for the ability to grow in the absence of tryptophan by streaking on
minimal plates lacking tryptophan. Some colonies are able to grow and some are not.
B). (4 points). What is the most likely explanation for each class of colonies? What phenotype
does this experiment select for?
The prototrophs are the result of excision at attL and attR restoring the integrity of the trpC
gene. The colonies that are still auxotrophs are the result of aberrant spontaneous excision
events. (These events could originate in the prophage and extend either leftward or
rightward or both. Some could leave behind some prophage genes if the deletion endpoint
is in the prophage. Some could extend into adjacent bacterial DNA). All of the survivors
must have the cI gene deleted so they can survive at 42o C.
