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

Chapter 3
3.6 Complementation of M. tuberculosis and M. smegmatis Cpn60.2 in ΔgroEL AI90
All the results so far have supported the understanding that Cpn60.2 is the main housekeeping
chaperonin of Mycobacteria. In this chapter it has been shown that Cpn60.2 can
function very well in E. coli MGM100 and Tab21. However, in all these experiments it was
possible that the P BAD promoter was a little leaky and that there was just enough GroEL
present within the cell, when grown on glucose, that it was capable of functioning along with
Cpn60.2 and sustaining cell growth. A lab colleague, Andrew Large, was able to show that
MGM100 cells grown on glucose did have very small amounts of residual GroEL by heavily
overloading SDS-PAGE gels and western blotting. So, to better test the hypothesis that
Cpn60.2 can fully complement in E. coli in the absence of endogenous GroEL, the
chromosomally encoded groEL gene would need to be removed from E. coli.
For this experiment the ΔgroEL strain, AI90, was used (Ivic et al., 1997). In this strain the
groES and groEL genes are present on the pACYC plasmid (pACYC-Ptrc-GroESL-SacB), as
it is not possible to delete the groEL gene without providing a complementing copy. It has
been shown that the sacB gene expression in gram negative bacteria like E. coli confers
sucrose sensitivity (Gay et al., 1985). When AI90 is grown on sucrose, it allows for the
selection of cells that no longer posses the sacB gene. In a lot of these cases, it is achieved
with the removal of the pACYC plasmid from the cells prior to the selection process. In such
a case, the AI90 strain would only grow if pACYC was displaced with a plasmid carrying
chaperonins capable of functioning in the complete absence of GroEL (plasmid shuffling). It
was expected that both M. tuberculosis and M. smegmatis Cpn60.2 would be able to rescue
cell growth in the complete absence of GroEL, as it had also been shown (T. Rao, PhD thesis)
that Cpn60.2 from M. smegmatis could complement in MGM100.
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