Part:BBa_K936014

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]