Team:Aachen/Project/FRET Reporter
From 2014.igem.org
(→References) |
AZimmermann (Talk | contribs) (→Achievements) |
||
Line 151: | Line 151: | ||
[[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]] | [[File:Aachen_14-10-15_Medal_Cellocks_iNB.png|right|150px]] | ||
- | == | + | == Results == |
<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. | ||
Line 166: | Line 168: | ||
Both double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 don't exhibit a strong fluorescence before induction with IPTG. In the non induced constructs the fluorescence stays low and only increases slightly over time. It is severely weaker than the fluorescence reached by the induced constructs or the positive control but also higher then the negative control. This shows that the promoter system used is not completely shut down without induction but significantly weaker compared with the induced constructs. | Both double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 don't exhibit a strong fluorescence before induction with IPTG. In the non induced constructs the fluorescence stays low and only increases slightly over time. It is severely weaker than the fluorescence reached by the induced constructs or the positive control but also higher then the negative control. This shows that the promoter system used is not completely shut down without induction but significantly weaker compared with the induced constructs. | ||
- | 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. | + | 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 K131 | ||
{{Team:Aachen/BlockSeparator}} | {{Team:Aachen/BlockSeparator}} |
Revision as of 16:22, 15 October 2014
|
|
|
|
|
|
|
|