Team:Aachen/Project/FRET Reporter
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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 = |
<span class="anchor" id="reachachievements"></span> | <span class="anchor" id="reachachievements"></span> | ||
- | + | ==Characterization of GFP-REACh1 and GFP-REACh 2 together with an IPTG inducible TEV protease== | |
The characterization of the TEV protease and the REACh 1 and REACh 2 dark quencher proteins was performed by introducing both simultaneously into ''E. coli''. The resulting double plasmid cells therefore contained [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP-REACh 1 fusion protein) and [http://parts.igem.org/Part:BBa_K1319008 K1319008] (IPTG inducible TEV protease) or [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP-REACh 2 fusion protein) and K1319008 respectively. K1319013 and K1319014 were situated on the pSB3K3 plasmid backbone and K1319008 on the pSB1C3 backbone, two standard plasmids with different oris allowing their simultaneous use in one cell. | The characterization of the TEV protease and the REACh 1 and REACh 2 dark quencher proteins was performed by introducing both simultaneously into ''E. coli''. The resulting double plasmid cells therefore contained [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP-REACh 1 fusion protein) and [http://parts.igem.org/Part:BBa_K1319008 K1319008] (IPTG inducible TEV protease) or [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP-REACh 2 fusion protein) and K1319008 respectively. K1319013 and K1319014 were situated on the pSB3K3 plasmid backbone and K1319008 on the pSB1C3 backbone, two standard plasmids with different oris allowing their simultaneous use in one cell. | ||
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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=== | |
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== | |
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+ | 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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+ | Therefore K731520 and the same double plasmid construct that was described earlier (K1319013 + K1319008 and K1319014 + K1319008) were cultivated in ''E. coli'' BL21(DE3) and fluorescence and OD were measured. Once again the fluorescence was adjusted for the OD to show a relative fluorescence on a cell per cell basis. Also it was especially looked at the difference between the induced and not induced state. This difference (fluorescence quotient) gives a better indicator for a system which is used as a sensor because the difference between an ''on'' and ''off'' state is more important for a clear and unmistakable signal compared to the overall fluorescence. Hence the OD adjusted fluorescence quotient for both double plasmid constructs (K1319013 + K1319008 and K1319014 + K1319008) and K731520 was obtained and plotted in the following graphic. | ||
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+ | '''''Add Florians graphic about K731520, K1319013 + K1319008 and K1319014 + K1319008 ''''' | ||
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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. | ||
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+ | ===summary=== | ||
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+ | The | ||
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{{Team:Aachen/BlockSeparator}} | {{Team:Aachen/BlockSeparator}} | ||
Revision as of 17:54, 15 October 2014
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