Composite

Part:BBa_K316008

Designed by: IC 2010 Team   Group: iGEM10_Imperial_College_London   (2010-10-21)
Revision as of 09:38, 27 October 2010 by Ks907 (Talk | contribs)

Cleavable GFP-XylE fusion with Pveg promoter and terminator

This part contains BBa_K316007 with added double terminator BBa_B0014. This construct is designed so that the XylE activity is substantially reduced untill such a time when a TEV protease is added to the system and transcribed. TEV protease cleavable linker is positioned between the two proteins. Once the linker is cleaved, XylE is free to tetramerise and assume full activity. GFP is His tagged at the 5' end to facilitate purificaiton for in-vitro assays. Terminator BBa_B0014 has been added to comply with Biobrick standards. This particular terminator is stronger and is different from BBa_B0015.

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].


Structure and Features

GFPXylETT.PNG

Figure I. Graphical representation of the GFP-XylE construct with associated Pveg promoter, tags, linkers and double terminator.


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 1229
    Illegal NgoMIV site found at 1401
    Illegal AgeI site found at 1752
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 785


Part Characterisation

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 relevant parts pages.

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