Difference between revisions of "Part:BBa K936021"
 
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LC Cutinase coding sequence with polyhistidine tag attached. The polyhistidine tag allows our research team to perform assays to discover where our enzyme is being released, both in the media and in the cell. <br><br>
 
LC Cutinase coding sequence with polyhistidine tag attached. The polyhistidine tag allows our research team to perform assays to discover where our enzyme is being released, both in the media and in the cell. <br><br>
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Through p-nitrophenyl butyrate (pNPB) assays, the UC Davis team gathered enough data to determine that the LC-Cutinase part (BBa_K936000) incorporated in this part most likely exhibits its intended function as an esterase. A detailed description of these assays can be found on the Module Engineering Project page: http://2012.igem.org/Team:UC_Davis/Project/Catalyst.  
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Through p-nitrophenyl butyrate (pNPB) assays, the UC Davis team gathered enough data to determine that the LC-Cutinase part (BBa_K936000) incorporated in this part most likely exhibits its intended function as an esterase. Additionally, the Cutinase part with a pelB tag (BBa_K936013) has been found to be secreted from the cells expressing it. A detailed description of these assays can be found on the Module Engineering Project page: http://2012.igem.org/Team:UC_Davis/Project/Catalyst.  
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<br>Additionally, the following data is further described on the UC Davis Cutinase Activity data page: http://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity
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https://static.igem.org/mediawiki/2012/a/aa/UC_Davis_First_pNPB_Run.png <br>(Above Graph) Results of pNPB assay detailing the constitutive construct as having the highest esterase activity.https://static.igem.org/mediawiki/2012/6/62/UC_Davis_Third_pNPB_Run.png<br>(Above Graphs) The graphs above detail higher activity among the constitutive construct and some mutant variants in esterase activity compared to the control and wild type per unit cell. <br>https://static.igem.org/mediawiki/2012/5/59/UC_Davis_Last_pNPB_Run.png<br> (Above graph) This graph represents a possible production of cutinase by the cells upon entering stationary phase.<br><br>
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https://static.igem.org/mediawiki/2012/d/d9/UCDavisParts2.png
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Western data of media samples probed for a his-tag shows that a protein of the length corresponding to cutinase (~30 kDa) is being secreted from the cell.
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<br><br>
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https://static.igem.org/mediawiki/2012/c/c4/UCDavisParts1.png
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<br>
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Quantitative measure of protein secretion into the media at different points after induction.
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<br><br>
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https://static.igem.org/mediawiki/2012/0/0b/UCDavisParts3.png https://static.igem.org/mediawiki/2012/6/63/UCDavisParts4.png https://static.igem.org/mediawiki/2012/2/28/UCDavisParts5.png
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The above plots show the results on pNPB assays in which esterase activity is measured by the absorbance at 405 nm. It is clearly shown that the activity of cells expressing cutinase is much higher the background (negative control).<br><br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 22:40, 13 October 2012

Cutinase and polyhistidine tag

LC Cutinase coding sequence with polyhistidine tag attached. The polyhistidine tag allows our research team to perform assays to discover where our enzyme is being released, both in the media and in the cell.

Through p-nitrophenyl butyrate (pNPB) assays, the UC Davis team gathered enough data to determine that the LC-Cutinase part (BBa_K936000) incorporated in this part most likely exhibits its intended function as an esterase. Additionally, the Cutinase part with a pelB tag (BBa_K936013) has been found to be secreted from the cells expressing it. A detailed description of these assays can be found on the Module Engineering Project page: http://2012.igem.org/Team:UC_Davis/Project/Catalyst.

UCDavisParts2.png
Western data of media samples probed for a his-tag shows that a protein of the length corresponding to cutinase (~30 kDa) is being secreted from the cell.

UCDavisParts1.png
Quantitative measure of protein secretion into the media at different points after induction.

UCDavisParts3.png UCDavisParts4.png UCDavisParts5.png
The above plots show the results on pNPB assays in which esterase activity is measured by the absorbance at 405 nm. It is clearly shown that the activity of cells expressing cutinase is much higher the background (negative control).

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 600
  • 1000
    COMPATIBLE WITH RFC[1000]