mohammad tabish ahmed - eTheses Repository - University of ...

Chapter 6
To ensure that the lack of complementation was not due to the lack of expression of Cpn60-
E458A, an SDS-PAGE gel was run (fig 6.6). Since Cpn60-E458A was causing cell death in
the presence of arabinose as well, it was difficult to induce the cells for long durations of
time. As a result, this plasmid could only be induced for 2-3 hours. It was observed that the
expression level of Cpn60-E458A was lower than that of Cpn60-K449G, Cpn60-G460A, and
Cpn60-V461A. However, even this level of expression should have been enough to allow
Cpn60-E458A to complement in MGM100 if it was functional.
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Fig 6.5: Complementation assay of Cpn60.2 mutants in glucose or arabinose. (a) Cells
grown on LB plates at 30°C with 0.2% arabinose and 0.1 mM IPTG. (b) Cells grown on LB
plates at 37°C with 0.2% glucose and 0.1 mM IPTG.
1- MGM100/ pTrc-GroES-Cpn60-K449G
2- MGM100/ pTrc-GroES-Cpn60-E458A
3- MGM100/ pTrc-GroES-Cpn60-G460A
4- MGM100/ pTrc-GroES-Cpn60-V461A
5- MGM100/ pTrc-GroES-GroEL
6- MGM100/ pTrc99a
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