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 [http://2010.igem.org/Team:Imperial_College_London/Results wiki results section]
The GFP-XylE fusion protein was tested in the K316007 pVEG based expression construct. The result can be seen on the experience page
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.
Structure and Features
Figure I. Graphical representation of the GFP-XylE construct with associated tags and linkers.
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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