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

Chapter 4
intermediate domains were from Cpn60.1 (referred to as PNR). However, as was the case
with DHF, the PCR product could not be cloned into the pTrc plasmid. No viable cells were
obtained after transformation into DH5α. It is known, from the experiments done with
Cpn60.2 in chapter 3, that the chaperonin constructs on the pTrc plasmid are induced at low
levels, even in the absence of IPTG. Since there was a possibility of this chimera being lethal
to the cells, even low levels of expression may have been causing cell death. As such,
attempts were made to better control its expression using the pBAD24 plasmid. The
expression of the chimera from this plasmid can be suppressed by growing the cells on
glucose media. Unfortunately, this method was also unsuccessful.
4.2.2 Chimeras JQL and PKR
Chimera JQL is the precise domain swap that has the equatorial and intermediate domain of
Cpn60.2 while the apical domain is from Cpn60.1. Chimera PKR has the reciprocal swap
with the equatorial and intermediate domains from Cpn60.1, while the apical domain is from
Cpn60.2. Both chimeras were made without any issues, and sequenced to ensure no
mutations were present. They were then transformed into MGM100 using the protocol
described in 2.4.16 to test their functional properties. The complementation method used was
unaltered from the previous experiments.
These swaps were made to see if incorporating the apical domain from Cpn60.2 into Cpn60.1
would allow it to function in E. coli, and if the reciprocal swap would prevent Cpn60.2 from
functioning. It was hypothesised that the apical domain of Cpn60.2 would allow Cpn60.1 to
recognise and bind to substrates that would rescue MGM100 growth, while the apical domain
of Cpn60.1 would prevent Cpn60.2 from functioning in its regular manner.
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