Part:BBa_K1362020

RBS + Xylanase

Xylanases are enzymes that degrad Xylan, a major component of wood. This is a modified version of BBa_K1175005 that is from B. subtilis (XylA). To ensure that it is kept intracellular we removed a signaling peptide from the N-terminus. Xylanases are interesting enzymes for industry as they have a huge variety of applications.

Activity after Heatshock assay for 30 minutes of linear Xylanase

Usage and Biology

To characterize our part in respect to its thermal stability we conducted a Heatshock Assay. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.

Fig. 3: Activity after Heatshock assay for 5 minutes of linear and circular Xylanase

In order to test the results with a biological replicate and to find out how a shorter heatshock influences the protein, we expressed the construct again and run another set of heatshock for 5 minutes at 63 °C. The data actually shows that with heatshock of 63 °C for the circular xylanase not all activity is lost but there is still some remaining activity. In contrast the assay confirms the initial data that the linear xylanase is not able to survive temperatures higher than 60 °C.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 22
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 426
  Illegal SapI.rc site found at 480