Part:BBa_K316006

N-terminus his tagged-GFP-XylE fusion protein

Constructed to be combined with promoter and terminator. The GFP BBa_E0040 has a 5'(5xHis) tag and is linked to XylE BBa_J33204 monomer subunit (It should be noted that the RBS of XylE (J33204) has been removed for this fusion protein). The linker is composed of a Flag tag (DYKDDDDK), TEV protease recognition sequence ENLYFQG followed by GGSGGS - flexible linker. The purpose of the linker and attached GFP is to render the XylE enzyme inactive, by preventing homo-tetramerization into its functional form.

For more information about XylE, it's substrate and spectrophotometric assays, please see BBa_K316003 or our wiki[http://2010.igem.org/Team:Imperial_College_London/Results]

Safety

The substrate XylE works on is a chemical called catechol. It is classed as irritant in the EU but as toxic in the USA, as well as being a possible carcinogen. It should therefore be handled with care and proper safety equipment. More information is available on the Material Safety Data Sheet[http://www.sciencelab.com/msds.php?msdsId=9927131].

Parts were assembled by PCR primer extension for exact methods, see our wiki [http://2010.igem.org/Team:Imperial_College_London/Strategy]

Structure and Features

Figure I. Graphical representation of the GFP-XylE construct with associated tags and linkers.

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 1106
  Illegal NgoMIV site found at 1278
  Illegal AgeI site found at 1629
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 662
  

For more information about our project please visit our wiki [http://2010.igem.org/Team:Imperial_College_London].