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.
Characterisation data was obtained using GFP-XylE constructs BBa_K316008 and XylE under two different promoters: B. subtilis derived Pveg BBa_K316005 and J23101 BBa_K316004 from E. coli. These are described on our wiki[http://2010.igem.org/Team:Imperial_College_London/Results] and the aforementioned parts pages.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1106
Illegal NgoMIV site found at 1278
Illegal AgeI site found at 1629 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 662
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