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

Chapter 4
4.1.5 Chimeras DHF & GEI
For these chimeras, attempts were made to make imprecise apical domain swaps between
GroEL and Cpn60.1. DHF is the chimera where the equatorial domain sequence and a partial
segment of the intermediate domain sequence are from Cpn60.1, while the apical domain
sequence and the remainder of the intermediate domain sequence come from GroEL. In GEI,
the equatorial domain sequence and a partial segment of the intermediate domain sequence
are from GroEL, while the apical domain sequence and the remainder of the intermediate
domain sequence come from Cpn60.1.
Unfortunately, as was the case with DBF in section 4.1.2, it was not possible to analyse the
functional and structural properties of DHF, as the final PCR product could not be cloned into
the pTrc plasmid. Attempts at transforming the ligated product into DH5α predominantly
caused cell death, while any colonies that were viable after the transformation process usually
had frameshift mutations in their chimera sequences. To try and resolve the frameshift issue,
site directed mutagenesis was performed using the Quickchange kit (Stratagene), as per the
manufacturer’s instructions, to reintroduce the missing nucleotide. However, no desirable
products were obtained. Again, it can only be speculated that the imprecise nature of the
domain swaps may have resulted in this chimera being lethal for the cells. As a result only the
clones with non functional chimera were viable.
Chimera GEI could not be analysed as no products were obtained after the final PCR. The
reasoning behind this is unclear, as the PCR process was largely successful up to this point.
Altering the protocol of the reaction by using a temperature gradient, increasing and reducing
extension times, altering the concentration of dNPTs used, and changing the ramping speed
had no effect. New primers were also used with no effect. As such, it was decided that the
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