Difference between revisions of "Part:BBa K2557002"
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==GFP-XylE expression in the presence or absence of TEV protease== | ==GFP-XylE expression in the presence or absence of TEV protease== | ||
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The graph presents the data acquired in an experiment to compare HMS production of cell cultures that: | The graph presents the data acquired in an experiment to compare HMS production of cell cultures that: | ||
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| − | 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 | + | 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 |
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Revision as of 13:16, 10 October 2018
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