Principle
The MIC value of chloramphenicol was determined by evaluating the growth of transformed WK6 cells on selective media. For that purpose, freshly transformed competent cells (i.e., cells obtained after the expression step in the transformation procedure - see section 4.1.3) were diluted 10- and 100-fold in LB medium, from which 20 µl fractions were spotted onto LB plates, containing chloramphenicol in concentrations ranging from 1 to 625 µg/ml.
The MIC value was defined as the minimal chloramphenicol concentration at which no growth had occurred after a 16 hours' incubation at 37°C. In all experiments, untransformed cells (MIC between 1 and 5 µg/ml) and pUCATI or pUCATIII transformed WK6, which produce the wild-type CAT variants, were included as references.
CAT mutants were defined to provide chloramphenicol resistance when their host grows on medium with standard chloramphenicol concentrations (i.e., at 25 mg/ml). CAT mutants that enable the growth of their host on media with 5 to 10 mg/ml chloramphenicol (but not with 25 µg/ml), were considered to be incompetent to confer chloramphenicol resistance, as a result of the former definition. These concentrations, however, are significantly higher than those on which untransformed WK6 cells can grow (2.5 mg/ml; Glasby, 1979). Hence, our definition of chloramphenicol resistance is different from the microbiological one.
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