Difference between revisions of "Part:BBa K2213003"
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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)_cgPPK2_mCherry_His6 (https://parts.igem.org/Part:BBa_K2213004).
 
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)_cgPPK2_mCherry_His6 (https://parts.igem.org/Part:BBa_K2213004).
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https://static.igem.org/mediawiki/2017/9/9c/Thermalshiftassaymanchesterjess3.png
 
https://static.igem.org/mediawiki/2017/9/9c/Thermalshiftassaymanchesterjess3.png
  
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Firgure 1: A thermal shift assay of the constructs BBa_K2213003 and BBa_K2213004. The data was normallised so that the highest reading of each run was equal to 1. Readings were taken at 0.4ºC intervals, 30 seconds after the solution had maintained that temperature.  
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Firgure 1: A thermal shift assay of the constructs BBa_K2213003 and BBa_K2213004 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 normalising the data, 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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From figure 1, cgPPK2His6 showed maximum change in denatured protein between 29.8ºC - 30.6ºC; whereas the tag-ppk-mcherry construct  showed peaks at 32.2ºC - 32.4ºC and
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 15:23, 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)_cgPPK2_mCherry_His6 (https://parts.igem.org/Part:BBa_K2213004).

Thermalshiftassaymanchesterjess3.png


Firgure 1: A thermal shift assay of the constructs BBa_K2213003 and BBa_K2213004 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 normalising the data, 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-ppk-mcherry construct showed peaks at 32.2ºC - 32.4ºC and

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