Team:Aachen/Project/FRET Reporter
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AZimmermann (Talk | contribs) (→Characterization of GFP-REACh1 and GFP-REACh 2 together with an IPTG inducible TEV protease) |
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[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]] | [[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]] | ||
- | = Achievements = | + | == Achievements == |
<span class="anchor" id="reachachievements"></span> | <span class="anchor" id="reachachievements"></span> | ||
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The induced double plasmid constructs exhibit a fast rise in fluorescence after induction up to an increase of over 10 fold compared to the non induced constructs. K1319013 + K1319008 reaches the the same level of fluorescence as I20260 indicating a complete cutting of the fusion proteins by the TEV protease. K1319014 + K1319008 doesn't reach the same level of fluorescence but the nearly 10 fold increase in fluorescence is a clear indicator for the TEV protease cutting the fusionprotein prodiced by K1319014. the same level of fluorescence as a the positive control is not achieved probably due to generally lower expression level of K1319014 in the cells. | The induced double plasmid constructs exhibit a fast rise in fluorescence after induction up to an increase of over 10 fold compared to the non induced constructs. K1319013 + K1319008 reaches the the same level of fluorescence as I20260 indicating a complete cutting of the fusion proteins by the TEV protease. K1319014 + K1319008 doesn't reach the same level of fluorescence but the nearly 10 fold increase in fluorescence is a clear indicator for the TEV protease cutting the fusionprotein prodiced by K1319014. the same level of fluorescence as a the positive control is not achieved probably due to generally lower expression level of K1319014 in the cells. | ||
- | ===Summary=== | + | ====Summary==== |
The double plasmid systems of K1319013 + K1319008 as well as K1319014 + K1319008 clearly demonstrate the Quenching ability of the REACh 1 and REACh 2 proteins as well as the funcionality of the TEV protease. Both REACH 1 and REACH 2 show a significant quenching ability of GFP shown in the difference of fluorescence between the positive control I20260 and the non induced double plasmid systems. This is also comfirmed by the resulting fluorescence after induction showing that the TEV protease is successfully able to cut the fusion proteins as well as the proper expression of both fusion proteins. Combined this characterization shows a validation of the functionality of the REACh 1 protein ([http://parts.igem.org/Part:BBa_K1319001 K1319001]), the REACh 2 protein ([http://parts.igem.org/Part:BBa_K1319002 K1319002]) and the TEV protease ([http://parts.igem.org/Part:BBa_K1319004 K1319004]). | The double plasmid systems of K1319013 + K1319008 as well as K1319014 + K1319008 clearly demonstrate the Quenching ability of the REACh 1 and REACh 2 proteins as well as the funcionality of the TEV protease. Both REACH 1 and REACH 2 show a significant quenching ability of GFP shown in the difference of fluorescence between the positive control I20260 and the non induced double plasmid systems. This is also comfirmed by the resulting fluorescence after induction showing that the TEV protease is successfully able to cut the fusion proteins as well as the proper expression of both fusion proteins. Combined this characterization shows a validation of the functionality of the REACh 1 protein ([http://parts.igem.org/Part:BBa_K1319001 K1319001]), the REACh 2 protein ([http://parts.igem.org/Part:BBa_K1319002 K1319002]) and the TEV protease ([http://parts.igem.org/Part:BBa_K1319004 K1319004]). | ||
- | ==Comparing the kinetic of the GFP-REACh fusion proteins with a standard lacI inducible GFP epression== | + | ===Comparing the kinetic of the GFP-REACh fusion proteins with a standard lacI inducible GFP epression=== |
To assess the kinetic of the fusion proteins K1319013 (GFP-REACh 1) and K1319014 (GFP-REACh 2) the double plasmid systems of K1319013 + K1319008 and K1319014 + K1319008 were compared to a standard expression of GFP under the control of a lacI promoter in [http://parts.igem.org/Part:BBa_K731520 K731520] made by the iGEM Team TRENTO in 2012 to evaluate the kinetic prediction of an a faster fluorescence response with our construct compared to a normal expression. | To assess the kinetic of the fusion proteins K1319013 (GFP-REACh 1) and K1319014 (GFP-REACh 2) the double plasmid systems of K1319013 + K1319008 and K1319014 + K1319008 were compared to a standard expression of GFP under the control of a lacI promoter in [http://parts.igem.org/Part:BBa_K731520 K731520] made by the iGEM Team TRENTO in 2012 to evaluate the kinetic prediction of an a faster fluorescence response with our construct compared to a normal expression. | ||
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The graphic clearly shows the faster kinetic of the cut GFP-REACh fusion protein compared to a standard GFP expression. Both fluorescence signals of the double plasmid constructs achieve a higher difference in fluorescence signal netween induced and non induced state as well as at a faster rate. This proves the earlier made hypothesis of the kinetic of the GFP-REACh fusion protein combined with a TEV protease. | The graphic clearly shows the faster kinetic of the cut GFP-REACh fusion protein compared to a standard GFP expression. Both fluorescence signals of the double plasmid constructs achieve a higher difference in fluorescence signal netween induced and non induced state as well as at a faster rate. This proves the earlier made hypothesis of the kinetic of the GFP-REACh fusion protein combined with a TEV protease. | ||
- | ===summary=== | + | ====summary==== |
The kinectic of the fusion protein combined with the TEV protease exhibits the exact characteristics as predicted earlier. The response is clearly faster than normal expression by accumulating a reservoir of fusion proteins who are not fluorescing due to the dark quencher attaches to it. This reservoir is then activated by the induction of the TEV protease which results in the cutting of the fusion protein, releasing GFP from the dark quencher and disturbing the FRET mechanims between it and GFP. This results in the observed faster fluorescence reaction due to multiplicating effect by the TEV protease in which every one TEV protease can account for many fluorescence proteins being activated. | The kinectic of the fusion protein combined with the TEV protease exhibits the exact characteristics as predicted earlier. The response is clearly faster than normal expression by accumulating a reservoir of fusion proteins who are not fluorescing due to the dark quencher attaches to it. This reservoir is then activated by the induction of the TEV protease which results in the cutting of the fusion protein, releasing GFP from the dark quencher and disturbing the FRET mechanims between it and GFP. This results in the observed faster fluorescence reaction due to multiplicating effect by the TEV protease in which every one TEV protease can account for many fluorescence proteins being activated. | ||
- | ==Characterizing the GFP-REACh constructs in [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor sensor chips]== | + | ===Characterizing the GFP-REACh constructs in [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor sensor chips]=== |
'''''Waiting for the Platereader data to be analyzed''''' | '''''Waiting for the Platereader data to be analyzed''''' |
Revision as of 13:35, 16 October 2014
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