We synthesized and cloned the Trigger Factor within a pUC18 plasmid system, under a Lac promoter operator. This plasmid system was chosen for its high copy number and efficient regulation of protein production by different IPTG concentrations. The protein expression analysis was carried out by running SDS PAGE, and the activity analysis was checked by transforming E. coli BL21(Rosetta), inducing growth by IPTG, and finally comparing the relative growth rates between different temperatures (18°C, 23°C and 37 °C). Encouragingly enough, we observed that the TF inclusion helped achieve a better growth of cells compared to non-insert samples.
We also cloned Cpn 60 into a pUC18 plasmid, and employed a Lac promoter operator system herein. Protein expression was checked by running SDS PAGE gels. The overall activity analysis was carried out as described above. Furthermore, since one of the bronze medal criteria is the characterization of existing parts, we decided to accomplish the same by carrying out a growth curve analysis of Cpn60 constructs under an IPTG inducible pLacI promoter at various temperatures. We’ve also achieved the co-expression of Cpn10/60 (polycistronic fashion) by combining both genes using sequential cloning. This was also placed under the Lac promoter operator system, and all other experimental and analytical assays were similar to those described above for the Cpn60 system.
