Difference between revisions of "Part:BBa K2213003"

Revision as of 18:33, 1 November 2017

cgPPK2_His6

Cornybacterium PPK2 with a C terminal polyhistidine tag, and the PduD(1-20) localisation tag at the N terminal

Usage and Biology

Characterisation

Assessing the thermal stability of cgPPK2 and PduD(1-20)-cgPPK2-mCherry
The heat stability of these constructs was determined through the use of a Thermal Shift Assay of the constructs cgPPK2_His6 (https://parts.igem.org/Part:BBa_K2213003) and PduD(1-20)_mCherry_cgPPK2_His6 (https://parts.igem.org/Part:BBa_K2213005).

Figure 1: A thermal shift assay of the constructs BBa_K2213003 and BBa_K2213005 using the dye sypro orange. Readings were taken at 0.4ºC intervals, 30 seconds after the solution had maintained that temperature. The samples were tested in duplicate before being normalised, so that the highest reading of each run was equal to 1; the mean was then plotted. Error bars showing the standard deviation are also shown.

From figure 1, cgPPK2His6 showed maximum change in denatured protein between 29.8ºC - 30.6ºC; whereas the tag-mcherry-ppk construct showed peaks at 32.2ºC - 32.4ºC. This suggests that the addition of a PduD tag and mCherry protein slightly increases the heat stability of the cgPPK2 protein. The tag-mCherry-PPK construct consistently showed a second peak at 95.2ºC, which is believed to be the mCherry domain unfolding.

Sequence and Features