1. Recently, the genome sequence of a Gram-negative bacterium Crazy bug 868 was reported. The gene abc is located in the chromosome of C. bug 868 and it encodes the enzyme ABC, which is involved in the biosynthesis of glutamic acid (E). You have isolated a spontaneous mutant C. bug 868-02 that cannot grow in chemically defined medium without supplementing glutamic acid (E-). You conducted an ABC enzyme activity assay and you found no activity in strain 868-02. With this experimental data in hand and your knowledge gained in molecular biology techniques:

A) Give two possible reasons why 868-02 is defective in ABC enzyme activity (i.e., where are the possible places for the mutation). 

B) If you believe it is because of a single mutation on the structural gene of abc, design an experiment to prove it? Outline your experimental approach including which cloning vector(s) you propose to use and what outcomes you expect. Assume: a) C. bug 868 is closely related to E. coli and all of the E.coli vectors work well in C. bug, b) the genomic sequence of C. bug 868 is available on the web and c) the DNA sequence of a cloned fragment can be determined using automated sequencing service.