Difference between revisions of "Part:BBa K2213003"
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Firgure 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.
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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.
 
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.

Revision as of 17:01, 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 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).

Thermalshiftassaymanchesterjess3.png


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 82
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 761