Team:Aachen/Project/FRET Reporter
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- | {{Team:Aachen/Figure|Aachen_Traditional_biosensor.png|align=center|title=Schematic model of a traditional biosensor|subtitle=In this model, the expression of GFP is directly controlled by a | + | {{Team:Aachen/Figure|Aachen_Traditional_biosensor.png|align=center|title=Schematic model of a traditional biosensor|subtitle=In this model, the expression of GFP is directly controlled by a promotor whose activator binds to a molecule secreted by the pathogen.|width=500px}} |
</center> | </center> | ||
- | As an alternative to the traditional approach, we '''constitutively express our reporter gene in a quenched form'''. in the GFP-REACh fusion protein fluorescence is suppressed. Our biosensor gives a response when homoserine lactones of ''P. aeruginosa'' are taken up by our sensor cells where the '''autoinducer activates the expression of the TEV protease''' by binding to the LasI | + | As an alternative to the traditional approach, we '''constitutively express our reporter gene in a quenched form'''. in the GFP-REACh fusion protein fluorescence is suppressed. Our biosensor gives a response when homoserine lactones of ''P. aeruginosa'' are taken up by our sensor cells where the '''autoinducer activates the expression of the TEV protease''' by binding to the LasI promotor in front of the protease gene. |
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<span class="anchor" id="gfp-reach"></span> | <span class="anchor" id="gfp-reach"></span> | ||
- | 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 [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319002 K1319002]. 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''', [http://parts.igem.org/Part:BBa_K1319016 K1319016]. As constitutive | + | 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 [http://parts.igem.org/Part:BBa_K1319001 K1319001] and [http://parts.igem.org/Part:BBa_K1319002 K1319002]. 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''', [http://parts.igem.org/Part:BBa_K1319016 K1319016]. As constitutive promotor 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 is 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] promotor''' that controls the expression of the TEV protease, which we make available as [http://parts.igem.org/Part:BBa_K1319004 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|align=center|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|align=center|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}} | ||
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- | {{Team:Aachen/Figure|align=center|Aachen 14-10-17 IPTG REACh iFG.png|title= Composition of our biosensor for IPTG|subtitle=To test the funtionality of our sensor concept, we expressed the TEV protease with a T7 | + | {{Team:Aachen/Figure|align=center|Aachen 14-10-17 IPTG REACh iFG.png|title= Composition of our biosensor for IPTG|subtitle=To test the funtionality of our sensor concept, we expressed the TEV protease with a T7 promotor.|width=900px}} |
{{Team:Aachen/BlockSeparator}} | {{Team:Aachen/BlockSeparator}} | ||
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The characterization of the TEV protease and the REACh1 and REACh2 dark quenchers was performed by introducing both simultaneously into ''E. coli'' Bl21 (DE3). The resulting double plasmid cells therefore contained [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP-REACh1 fusion protein) or [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP-REACh2 fusion protein) and [http://parts.igem.org/Part:BBa_K1319008 K1319008] (IPTG-inducible TEV protease). K1319013 and K1319014 were located on a pSB3K3 plasmid backbone and K1319008 on a pSB1C3 backbone, two standard iGEM plasmids with different oris allowing simultaneous use in one cell. | The characterization of the TEV protease and the REACh1 and REACh2 dark quenchers was performed by introducing both simultaneously into ''E. coli'' Bl21 (DE3). The resulting double plasmid cells therefore contained [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP-REACh1 fusion protein) or [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP-REACh2 fusion protein) and [http://parts.igem.org/Part:BBa_K1319008 K1319008] (IPTG-inducible TEV protease). K1319013 and K1319014 were located on a pSB3K3 plasmid backbone and K1319008 on a pSB1C3 backbone, two standard iGEM plasmids with different oris allowing simultaneous use in one cell. | ||
- | [http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because I20260 contains the same | + | [http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because I20260 contains the same promotor ([http://parts.igem.org/Part:BBa_J23101 J23101]), the same RBS ([http://parts.igem.org/Part:BBa_B0032 B0032]) and the same version of GFP ([http://parts.igem.org/Part:BBa_E0040 E0040]) and is located on the same plasmid backbone pSB3K3. Therefore, it is expected that when all fusion proteins are successfully cut by the TEV protease, the fluorescence level of the double plasmid constructs reaches the same level as the positive control of I20260. As a negative control [http://parts.igem.org/Part:BBa_B0015 B0015] was used, a coding sequence of a terminator which should not show any sign of fluorescence. |
To characterize our REACh1/2 constructs in combination with the TEV protease a growth experiment was conducted. Both of the double plasmid constructs, a constitutive expression of GFP (I20260) as positive control and B0015 as negative control were compared. For each expression IPTG-induced and non-induced cultures were grown in parallel. All measurements were done in a biological triplicate. | To characterize our REACh1/2 constructs in combination with the TEV protease a growth experiment was conducted. Both of the double plasmid constructs, a constitutive expression of GFP (I20260) as positive control and B0015 as negative control were compared. For each expression IPTG-induced and non-induced cultures were grown in parallel. All measurements were done in a biological triplicate. | ||
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The negative control B0015 did not exhibit any significant fluorescence. The positive control I20260 showed a steady level of fluorescence as expected due to the constitutive expression of GFP. As expected, the production is also independent of addition of IPTG therefore the triplicates have been merged together. | The negative control B0015 did not exhibit any significant fluorescence. The positive control I20260 showed a steady level of fluorescence as expected due to the constitutive expression of GFP. As expected, the production is also independent of addition of IPTG therefore the triplicates have been merged together. | ||
- | Both double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 did not exhibit a strong fluorescence before induction with IPTG. In the non-induced state, the fluorescence stays low and does not increase over time. It is significantly weaker than the fluorescence reached by the induced constructs or the positive control but was higher than the negative control. The higher base level of fluorescence in the not induced constructs is due to the imperfect quenching and the leakiness of the IPTG inducible | + | Both double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 did not exhibit a strong fluorescence before induction with IPTG. In the non-induced state, the fluorescence stays low and does not increase over time. It is significantly weaker than the fluorescence reached by the induced constructs or the positive control but was higher than the negative control. The higher base level of fluorescence in the not induced constructs is due to the imperfect quenching and the leakiness of the IPTG inducible promotor. |
The induced double plasmid constructs exhibited a fast rise in fluorescence after induction. '''The signal strenght increased ~ 10-fold over the non-induced constructs.''' K1319014 + K1319008 reached the the same level of fluorescence as I20260, indicating a complete cleavage of the fusion proteins by the TEV protease. K1319013 + K1319008 did not reach a level of fluorescence as high as K1319013 + K1319008, however, the nearly 10-fold increase in fluorescence after induction is a clear indicator for the TEV protease cutting the fusion protein K1319013. The weaker fluorescence signal is probably due to a lower expression level of K1319013 in the cells compared to the expression level of K1319014. | The induced double plasmid constructs exhibited a fast rise in fluorescence after induction. '''The signal strenght increased ~ 10-fold over the non-induced constructs.''' K1319014 + K1319008 reached the the same level of fluorescence as I20260, indicating a complete cleavage of the fusion proteins by the TEV protease. K1319013 + K1319008 did not reach a level of fluorescence as high as K1319013 + K1319008, however, the nearly 10-fold increase in fluorescence after induction is a clear indicator for the TEV protease cutting the fusion protein K1319013. The weaker fluorescence signal is probably due to a lower expression level of K1319013 in the cells compared to the expression level of K1319014. |
Revision as of 23:17, 17 October 2014
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