Coding

Part:BBa_K316003

Designed by: IC 2010 Team   Group: iGEM10_Imperial_College_London   (2010-10-22)
Revision as of 06:46, 27 October 2010 by Ks907 (Talk | contribs)

XylE - catechol 2,3-dioxygenase from P.putida with terminator

Catechol or catechol 2,3-dioxygenases + O(2) is converted by a ring cleavage into 2-hydroxymuconate semialdehyde which is the toxic and bright yellow-coloured substrate1. This is a key enzyme in many (soil) bacterial species used for the degradation of aromatic compounds. Catechol 2,3-dioxygenase2 was originally isolated from Pseudomonas putida and is a homotetramer of C230 monomers. The tetramerization interactions position a ferrous ion critical for enzymatic activity. It has been deduced that intersubunit interaction is essential to produce a functioning enzyme after performing N and C terminal modifications on the monomer. Coming together the subunits generate an active site. The reaction itself takes place within seconds after the addition by Pasteur pipette or spraying of catechol at a 100mM stock solution diluted with DDH20 (used by our lab.) The toxic byproduct is thought to interfere with cell wall integrity and cellular machinery such that exposed cells gradually die.


Safety

Catechol 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 from MSDS document[http://www.sciencelab.com/msds.php?msdsId=9927131].



Please see ‘Part Design’ section for design considerations and parts used.


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 337
    Illegal NgoMIV site found at 509
    Illegal AgeI site found at 860
  • 1000
    COMPATIBLE WITH RFC[1000]


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 in our wiki[http://2010.igem.org/Team:Imperial_College_London/Results] and the relevant parts pages.


References

<biblio>

  1. 1 pmid=10368270
  2. 2 pmid=12519074
  3. 3 pmid=6405380

</biblio>

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Parameters
n/aXylE - catechol 2,3-dioxygenase from P.putida with terminator