Difference between revisions of "Part:BBa K316006"

(→‎Structure and Features)
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Constructed to be combined with promoter and terminator. The GFP <bbpart>BBa_E0040</bbpart> has a 5'(5xHis) tag and is linked to XylE <bbpart>BBa_J33204</bbpart> monomer subunit (It should be noted that the RBS of XylE (<bbpart>J33204</bbpart>) 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.
 
Constructed to be combined with promoter and terminator. The GFP <bbpart>BBa_E0040</bbpart> has a 5'(5xHis) tag and is linked to XylE <bbpart>BBa_J33204</bbpart> monomer subunit (It should be noted that the RBS of XylE (<bbpart>J33204</bbpart>) 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.
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'''Safety'''
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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].
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Revision as of 06:50, 27 October 2010

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.


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.


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].