Difference between revisions of "Part:BBa K2213003"
| Line 23: | Line 23: | ||
</center> | </center> | ||
<b>Figure 2:</b> SDS-PAGE analysis of the different stages of purification for the cgPPK construct | <b>Figure 2:</b> SDS-PAGE analysis of the different stages of purification for the cgPPK construct | ||
| − | |||
<br> | <br> | ||
Revision as of 22:32, 1 November 2017
cgPPK2_His6
Cornybacterium glutamicum class II polyphosphate kinase (cgPPK) with a fused C terminal hexa-histidine tag to allow purification using immobilised metal ion affinity chromatography. First isolated and characterised by Lindner et al., (2007). Under the experimental conditions used by the authors, cgPPK was found to have a specific activity of 30/s. Under our own experimental conditions
Figure 1. Comparison of specific activity between the E. coli class I PPK and the Corynebacterium glutamicum class II PPK
Lindner, S., Vidaurre, D., Willbold, S., Schoberth, S. and Wendisch, V. (2007). NCgl2620 Encodes a Class II Polyphosphate Kinase in Corynebacterium glutamicum. Applied and Environmental Microbiology, 73(15), pp.5026-5033.
Usage and Biology
Characterisation
The part was over expressed in a BL21 (DE3) strain of E. coli and successfully purified to near homogeneity using Ni-NTA affinity media (Figure 2).
Figure 2: SDS-PAGE analysis of the different stages of purification for the cgPPK construct
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 3: 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 3, 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 also consistently showed a second peak at 95.2ºC. Using Imperial2011's experience with BBa_I13521 (https://parts.igem.org/Part:BBa_I13521:Experience) we believe this peak is likely the mCherry domain unfolding.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 82
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 761
/