Composite
GFP-XylE

Part:BBa_K316006

Designed by: IC 2010 Team   Group: iGEM10_Imperial_College_London   (2010-10-21)
Revision as of 00:45, 28 October 2010 by Ks907 (Talk | contribs)

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]

The GFP-XylE fusion protein was tested in the K316007 pVEG based expression construct. The result can be seen on the experience page


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

NaGFPXylE.PNG


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


Characterisation data was obtained for XylE BBa_K316003. In addition constructs under two different promoters: J23101-XylE BBa_K316004 from E. coli was used to categorise B. subtilis derived Pveg-XylE BBa_K316005. Also GFP-XylE constructs BBa_K316007 were tested to determine the effectiveness of inhibition of XylE activity by attachment of GFP. These are described on our wiki[http://2010.igem.org/Team:Imperial_College_London/Results] and the aforementioned parts pages.


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


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