Difference between revisions of "Part:BBa K936020"
 
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Full Cutinase Construct on psB1A3 backbone. <br>
 
Full Cutinase Construct on psB1A3 backbone. <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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<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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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>
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<br>
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https://static.igem.org/mediawiki/2012/d/d9/UCDavisParts2.png
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<br>
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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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<br>
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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).
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===Usage and Biology===
 
===Usage and Biology===
  
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<br>Group: Harvard Biodesign 2016<br>
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Author: Berner, Gabi; Chun, Rebekah; Edun, Mofeyifoluwa <br>
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Summary: By histag purification using a nickel column, we were able to purify LC-Cutinase and determine that it was secreted into the extracellular medium of the induced BL21 cells.  This potentially addresses issues with purifying the protein. More detailed information on our methods is available here: http://2016.igem.org/Team:Harvard_BioDesign/Experiments<br>
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<a href="https://static.igem.org/mediawiki/parts/6/62/BBaK936020_Western.jpeg"><img src="https://static.igem.org/mediawiki/parts/6/62/BBaK936020_Western.jpeg" style="width:600px;margin-left:180px"  ></a>
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<p style="text-align:center">Figure.  Western Blot of LC Cutinase Expressed in BL21 E. Coli</p>
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The western blot shows that the enzyme came out in the flow-through and eluted at an imidazole concentration of 10mM. Usually this elute is used to remove proteins that bind non-specifically to the nickel column. However, the fact that the band is present on the Western Blot suggests that the his tag is able to bind specifically.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 01:12, 20 October 2016

K206000+B0034+pelB+Cutinase+Polyhistidine Tag

Full Cutinase Construct on psB1A3 backbone.


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).

Usage and Biology


Group: Harvard Biodesign 2016
Author: Berner, Gabi; Chun, Rebekah; Edun, Mofeyifoluwa
Summary: By histag purification using a nickel column, we were able to purify LC-Cutinase and determine that it was secreted into the extracellular medium of the induced BL21 cells. This potentially addresses issues with purifying the protein. More detailed information on our methods is available here: http://2016.igem.org/Team:Harvard_BioDesign/Experiments


Figure. Western Blot of LC Cutinase Expressed in BL21 E. Coli


The western blot shows that the enzyme came out in the flow-through and eluted at an imidazole concentration of 10mM. Usually this elute is used to remove proteins that bind non-specifically to the nickel column. However, the fact that the band is present on the Western Blot suggests that the his tag is able to bind specifically.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 210
    Illegal AgeI site found at 822
  • 1000
    COMPATIBLE WITH RFC[1000]