Difference between revisions of "Part:BBa K936014"

Latest revision as of 00:33, 27 October 2012

pelB-LC-Cutinase with polyhistidine-tag

pelB leader sequence attached to enzyme LC-Cutinase protein coding sequence in order to allow the cell to bring the enzyme to the periplasm for easier secretion. The polyhistidine-tag allows for us to purify the produced enzyme.
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.

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.

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

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 NgoMIV site found at 54
Illegal AgeI site found at 666
1000
COMPATIBLE WITH RFC[1000]

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 pelB leader sequence attached to enzyme LC-Cutinase protein coding sequence in order to allow the cell to bring the enzyme to the periplasm for easier secretion. The polyhistidine-tag allows for us to purify the produced enzyme. <br>
-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>
+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.
+<br>
+<br>
+https://static.igem.org/mediawiki/2012/d/d9/UCDavisParts2.png
+<br>
+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.
+<br><br>
+https://static.igem.org/mediawiki/2012/c/c4/UCDavisParts1.png
+<br>
+Quantitative measure of protein secretion into the media at different points after induction.
+<br><br>
+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
+<br>
+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).
 <!-- Add more about the biology of this part here