Difference between revisions of "Part:BBa K1362022"

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Xylanases are enzymes that degrad Xylan, a major component of wood. This is the circular counterpart of <partinfo>BBa_K1362020</partinfo>. A circular Xylanase should be resistent to exo-peptidases and could be more heat stable because of its reduced conformational space. This could be interesting for industrial applications, as they are often performed at higher temperatures and harsher environments.
 
Xylanases are enzymes that degrad Xylan, a major component of wood. This is the circular counterpart of <partinfo>BBa_K1362020</partinfo>. A circular Xylanase should be resistent to exo-peptidases and could be more heat stable because of its reduced conformational space. This could be interesting for industrial applications, as they are often performed at higher temperatures and harsher environments.
  
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[[File:Heatshockassay_circular_nonpurified_xylanase.png|300px|thumb|right|Activity after Heatshock assay for 30 minutes of circular Xylanase]]
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===Usage and Biology===
 
===Usage and Biology===
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[[File:XylanaseCoomassieCircularizationShift.png|440px|thumb|right|Fig. 1: SDS PaGe indicating successful circularization]]
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The first interesting question for the circular xylanase is did the circulrization was actually successful. We therefore made a SDS Polyacrylamide Gel stained with Coomassie Billiant Blue. It shows a visible sift between the linear and the circular construct as the circular is running a little bit faster due to increased electrophoretic mobility (Fig. 1)
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[[File:Heatshockassay_circular_nonpurified_xylanase.png|300px|thumb|right|Activity after Heatshock assay for 30 minutes of circular Xylanase]]
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[[File:MainStabilityResult.png|440px|thumb|right|Fig. 3: Activity after Heatshock assay for 5 minutes of linear and circular Xylanase]]
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To characterize our part in respect to its thermal stability we conducted a Heatshock Assay. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.
 
To characterize our part in respect to its thermal stability we conducted a Heatshock Assay. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.
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In order to test the results with a biological replicate and to find out how a shorter heatshock influences the protein, we expressed the construct again and run another set of heatshock for 5 minutes at 63 °C. The data actually shows that with heatshock of 63 °C for the circular xylanase not all activity is lost but there is still some remaining activity. In contrast the assay confirms the initial data that the linear xylanase is not able to survive temperatures higher than 60 °C.
  
  

Latest revision as of 04:39, 2 November 2014

RBS + Circular Xylanase

Xylanases are enzymes that degrad Xylan, a major component of wood. This is the circular counterpart of BBa_K1362020. A circular Xylanase should be resistent to exo-peptidases and could be more heat stable because of its reduced conformational space. This could be interesting for industrial applications, as they are often performed at higher temperatures and harsher environments.


Usage and Biology

Fig. 1: SDS PaGe indicating successful circularization

The first interesting question for the circular xylanase is did the circulrization was actually successful. We therefore made a SDS Polyacrylamide Gel stained with Coomassie Billiant Blue. It shows a visible sift between the linear and the circular construct as the circular is running a little bit faster due to increased electrophoretic mobility (Fig. 1)

Activity after Heatshock assay for 30 minutes of circular Xylanase
Fig. 3: Activity after Heatshock assay for 5 minutes of linear and circular Xylanase

To characterize our part in respect to its thermal stability we conducted a Heatshock Assay. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.

In order to test the results with a biological replicate and to find out how a shorter heatshock influences the protein, we expressed the construct again and run another set of heatshock for 5 minutes at 63 °C. The data actually shows that with heatshock of 63 °C for the circular xylanase not all activity is lost but there is still some remaining activity. In contrast the assay confirms the initial data that the linear xylanase is not able to survive temperatures higher than 60 °C.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 153
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 557
    Illegal SapI.rc site found at 611