Team:Aachen/Project/FRET Reporter
From 2014.igem.org
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==Producing a GFP-REACh Fusion Protein== | ==Producing a GFP-REACh Fusion Protein== | ||
- | In our project, we reproduced the REACh1 and REACh2 proteins by subjecting an RFC-25 compatible version of the BioBrick [http://parts.igem.org/Part:BBa_E0030 E0030] (EYFP) to a '''QuikChange mutation''', creating the BioBricks K1319001 and K1319002, respectively. Subsequently, we fused each REACh protein with '''GFP (mut3b)''' which is available as BioBrick [http://parts.igem.org/Part:BBa_E0040 E0040]. The protein complex was linked via a '''protease cleavage site''', K1319016. | + | In our project, we reproduced the REACh1 and REACh2 proteins by subjecting an RFC-25 compatible version of the BioBrick [http://parts.igem.org/Part:BBa_E0030 E0030] (EYFP) to a '''QuikChange mutation''', creating the BioBricks K1319001 and K1319002, respectively. Subsequently, we fused each REACh protein with '''GFP (mut3b)''' which is available as BioBrick [http://parts.igem.org/Part:BBa_E0040 E0040]. The protein complex was linked via a '''protease cleavage site''', K1319016. As constitutive promoter we use [http://parts.igem.org/Part:BBa_J23101 J232101]. When GFP is connected to either REACh quencher, GFP will absorb light but the energy will be transferred to REACh via FRET and then emitted in the form of heat; the fluorescence is quenched. Our cells also constitutively express the '''[http://parts.igem.org/Part:BBa_C0179 LasR] activator'''. Together with the HSL molecules from ''P. aeruginosa'', LasR binds to the '''[http://parts.igem.org/Part:BBa_J64010 LasI] promoter''' that controls the expression of the TEV protease, which we make available as K1319004. When the fusion protein is cleaved by the TEV protease, REACh will be separated from GFP. The latter will then be able to absorb and emit light as usual. |
{{Team:Aachen/Figure|Aachen_14-10-08_REACh_approach_with_BioBricks_iNB.png|title=Composition of our biosensor|subtitle=For our biosensor, we use a mix of already available and self-constructed BioBricks.|width=900px}} | {{Team:Aachen/Figure|Aachen_14-10-08_REACh_approach_with_BioBricks_iNB.png|title=Composition of our biosensor|subtitle=For our biosensor, we use a mix of already available and self-constructed BioBricks.|width=900px}} |
Revision as of 09:40, 10 October 2014
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