A trio of APuS genetics students created a mutant strain of E. col that, unlike

Question

A trio of APuS genetics students created a mutant strain of E. col that, unlike the normal strain, cannot synthesize methionine (I.e. it is auxotrophic for MET). MET therefore must be present in its growth medium. This is kind of a big deal, since MET corresponds to the stert codon in DNA transcription. Each of the 3 students bulit plasmid DNA libraries from the wild-type parental (normal) strain. They each used different restriction enzymes to clone the fragments, and then transformed the mutant (auxotrophic) strain with their own library. Their chosen restriction enzyme and their results are as follows Student 1: used EcoRI; lots of prototrophic colonles in very littie medium Student 2: used HINDIII no colonies Student 3: Xhol; no colonies Their experimental control suggests that the actual transformation process worked fine, so how can these resuits be explained? (HINT: Think about how restriction enzymes work, their degree of precision, etc.)

Explanation / Answer

It is clear that the mutant gene that inhibits the synthesis of MET is active in the transformed strains of students 2 and 3. Because it is known that Met is the start codon and is essential that the genetic machinery reads that to start translation. But if a mutant strain is unable to read MET, no proteins will be translated and hence no colonies will be formed.

However, since restriction enzymes are known to cleave at a particular site in the genome, it may happen that the mutant gene is cleaved by EcoRI and has become non functional. As a result this transformed strain can read the code for MET and also synthesise it. And since it can read MET, the translation will begin and proteins will be formed. And also MET will be synthesised and we will obtain prototrophc colonies.

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