Part:BBa_K2557002

Bxb1 attB-RFP-Bxb1 attP

We added Bxb1 recombination sites, attB and attP, at both ends of the reporter gene RFP (Inverted). When Bxb1 is expressed, attB and attP are recognized, and the sequence of RFP is inverted to enable normal expression.

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 [http://www.sciencelab.com/msds.php?msdsId=9927131 Material Safety Data Sheet].

Parts were assembled by PCR primer extension for exact methods, see our [http://2010.igem.org/Team:Imperial_College_London/Strategy wiki assembly strategy]

GFP-XylE expression in the presence or absence of TEV protease

The graph presents the data acquired in an experiment to compare HMS production of cell cultures that:

1)Express XylE gene (blue)

2)Express GFP-XylE in the absence of TEV (red)

3)Express GFP-XylE along with TEV protease (green)

The results show that the GFP-XylE fusion is far less able to tetramerise to form active XylE enzymes and break down catechol. This is shown by the difference between the rates of catechol breakdown (measured by increasing optical density at 380nm caused by accumulation of the breakdown product 2hydroxymuconic semi-aldehyde - (HMS)) between XylE alone (blue) and GFP-XylE in the absence of TEV (red). TEV protease (expressed in the cell with the GFP-XylE) cleaves the GFP-XylE, producing active XylE protein. This can be seen by comparing the blue (XylE) and the green (GFP-XylE in presence of TEV) curve data, as they show similar rates of production of HMS colored product.

The plateauing effect is caused by depletion of the catechol substrate and differences in the height of the absorbance plateaus value is due to slightly different initial concentrations of catechol substrate (both approximately 0.2mM).

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.

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Structure and Features

<img src="" width="800px">

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

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 837
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 36
  Illegal BsaI site found at 177
  Illegal BsaI.rc site found at 885
  Illegal SapI site found at 794