Difference between revisions of "Part:BBa K936020"

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.

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).

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]

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-The above gel 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.
+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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