Team:Aachen/Project/FRET Reporter

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{{Team:Aachen/Header}}
{{Team:Aachen/Header}}
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=The REACh Construct=
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=The modified REACh Construct=
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On this page, we present our biosensor on the molecular level. Explore the different parts of our genetic device:
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On this page, we present our biosensor on a molecular level. Here you can explore the different parts of our genetic device:
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<ul class="menusmall-grid" style="width:inherit;">
<!-- Overview -->
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     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#fluorescence" style="color:black">
     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#fluorescence" style="color:black">
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     <div class="menusmall-item menusmall-info" ><div class="menukachel">A Faster Answer</div></div>
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     <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">A Faster Answer</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0b/Aachen_14-10-13_GFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/0b/Aachen_14-10-13_GFP_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
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     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#fret" style="color:black">
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     <div class="menusmall-item menusmall-info" ><div class="menukachel">The FRET System</div></div>
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     <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">The FRET System</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/54/Aachen_14-10-13_FRET_Arrows_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/5/54/Aachen_14-10-13_FRET_Arrows_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
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     <div class="menusmall-item menusmall-info" ><div class="menukachel">REACh Quenchers</div></div>
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     <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">REACh Quenchers</div></div>
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     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/65/Aachen_14-10-13_REACh_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
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     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#gfp-reach" style="color:black">
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     <div class="menusmall-item menusmall-info" ><div class="menukachel">The Fusion Protein</div></div>
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     <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">The Fusion Protein</div></div>
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/02/Aachen_14-10-13_Fusion_Protein_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
     <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/0/02/Aachen_14-10-13_Fusion_Protein_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
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     <div class="menusmall-item menusmall-info" ><div class="menukachel">TEV Protease</div></div>
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     <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">TEV Protease</div></div>
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     <a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#reachachievements" style="color:black">
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     <div class="menusmall-item menusmall-info" ><div class="menukachel">Achieve-<br/>ments</div></div>
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     <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:32%; line-height:1.5em;">Achieve-<br/>ments</div></div>
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    <a class="menulink" href="https://2014.igem.org/Team:Aachen/Project/FRET_Reporter#reachoutlook" style="color:black">
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    <div class="menusmall-item menusmall-info" ><div class="menukachel" style="top:40%;">Outlook</div></div>
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    <div class="menusmall-item menusmall-img" style="background: url(https://static.igem.org/mediawiki/2014/6/67/Aachen_14-10-16_Outlook_Cellocks_iNB.png); norepeat scroll 0% 0% transparent; background-size:100%">
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[[File:Aachen_14-10-13_GFP_iNB.png|150px|right]]
[[File:Aachen_14-10-13_GFP_iNB.png|150px|right]]
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= A Fluorescence Answer Faster Than Expression =
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= A Fluorescence Answer Faster than Expression =
<span class="anchor" id="fluorescence"></span>
<span class="anchor" id="fluorescence"></span>
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Biosensors often work with a system that is comprised of a reported gene under the control of a promoter that is induced directly by the chemical that the sensor is supposed to detect. In the case of our 2D biosensor for ''Pseudomonas aeruginosa'', the expression of our reporter gene, GFP, would be directly induced by the activity of the bacterium's quorum sensing molecules. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we wanted to use a system that gives a fluorescent answer fast than just expressing the fluorescent protein.
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Biosensor systems are usually based on a reporter gene under the control of a promoter directly induced by the biomolecule of interest. In the case of our 2D biosensor for ''Pseudomonas&nbsp;aeruginosa'', the expression of our fluorescing reporter protein is directly induced by the activity of the bacterium's quorum sensing molecules. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we aimed to develop a system that provides a fluorescent answer faster than just expressing the fluorescent protein GFP.
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{{Team:Aachen/Figure|Aachen_Traditional_biosensor.png|align=center|title=Approach of a traditional biosensor|subtitle=In this model, the expression of GFP is directly controlled by a promoter whose activator binds to a molecule secreted by the pathogen.|width=500px}}
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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 promoter whose activator binds to a molecule secreted by the pathogen.|width=500px}}
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Instead of the traditional approach, we '''constitutively express our reporter gene in its quenched form'''. As GFP_REACh fusion protein, fluorescence is suppressed. Our biosensor gives a response when homoserine lactones of ''Pseudomonas aeruginosa'' are taken up by our sensor cells where the '''autoinducer activates the expression of the TEV protease''' by binding to the LasI promoter in front of the protease gene.
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As an alternative to the traditional approach, we '''constitutively express our reporter gene in a quenched form'''. By fusing GFP to REACh, a mutated variant of EYFP, its original fluorescence is thoroughly suppressed. Thereby, a huge stock of temporary inactivated fluorescence proteins is already available prior to first contact with the pathogen. As a conseqence to the uptake of homoserine lactones of ''P.&nbsp;aeruginosa'' and binding to the LasI promoter in front of the protease gene, these '''autoinducers activate the expression of the TEV protease'''. This way, our biosensor provides a fast increase in fluorescence intensity by cleavage of GFP-REACh-constructs.
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{{Team:Aachen/Figure|Aachen_REACh_approach.png|align=center|title=Our novel biosensor approach|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elicit a fluorescence response.|width=900px}}
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{{Team:Aachen/Figure|Aachen_REACh_approach.png|align=center|title=Schematic model of our novel biosensor|subtitle=Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elicit a fluorescence response.|width=900px}}
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This approach has two advantages:
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'''Advantages of the novel biosensor:'''
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* When ''Pseudomonas aeruginosa'' is detected by our cells, the reporter protein is already expressed and only waits to be cleaved off the REACh quencher. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. This way we hope for '''an earlier response''' by our sensor cells.
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* While a certain concentration of homoserine lactone will produce the same number of gene read-outs, one TEV protease can cleave many GFP_REACh constructs. Through the cleavage step we therefore introduce an '''amplification step''' into our system. With the TEV protease, we will be able to produce '''a much stronger signal''' in a short time interval.
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* When ''P.&nbsp;aeruginosa'' is detected by our cells, the reporter protein has already been expressed and only waits to be activated. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. Therefore, our sensor cells provide an exceptionally '''early response'''.
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<center>
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* While a certain concentration of homoserine lactone will produce the same number of gene read-outs and the TEV protease is about the same size as GFP (~27 kDa), one TEV protease can cleave many GFP-REACh constructs per time than fluorescent protein molecules could be expressed and go through fluorescence maturation. Through this additional reaction, we introduce an '''amplification step''' into our system. Hypothetically, the TEV protease will enable the production of '''a much stronger signal''' in a given time interval.
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{{Team:Aachen/Figure|Aachen_Model_merged.png|align=center|title=|width=1000px}}
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In parallel, we worked on a [https://2014.igem.org/Team:Aachen/Project/Model model] of the respective biosensor system, in order to confirm our hypothesis of a faster and stronger fluorescence response than a conventional biosensor would povide.
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The novel biosensor approach was modeled as shown above. The plotted results also include a model of a direct expression of GFP as it appears in traditional biosensors. The strength of the promotor used for the traditional approach is twice as high as the strength of the promotor upstream of the TEV coding sequence in the novel approach. Despite the stronger promotor a higher GFP concentration is generated in the model of the novel biosensor proving the stronger and faster output of our construct in theory.
 
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Förster resonance energy transfer (FRET), sometimes also called  fluorescence resonance energy transfer, is a physical process of energy transfer. In FRET, the energy of a donor chromophore, whose electrons are in an excited state, is passed to a second chromophore, the acceptor. The '''energy is transferred without radiation''' and is therefore not exchanged via emission and absorption of photons. The acceptor then releases the energy received from the donor, for example, as light of a longer wavelength.  
Förster resonance energy transfer (FRET), sometimes also called  fluorescence resonance energy transfer, is a physical process of energy transfer. In FRET, the energy of a donor chromophore, whose electrons are in an excited state, is passed to a second chromophore, the acceptor. The '''energy is transferred without radiation''' and is therefore not exchanged via emission and absorption of photons. The acceptor then releases the energy received from the donor, for example, as light of a longer wavelength.  
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In biochemistry and cell biology, fluorescent dyes, which interact via FRET, are applied as "optical nano metering rules", because the intensity of the transfer is dependend on the spacing between donor and acceptor, and can be observed up to distances of 10&nbsp;nm. This way, protein-protein interactions and conformational changes of a variety of tagged biomolecules can be observed. For efficient FRET to occur, there must be a substantial overlap between the donor fluorescence emission spectrum and the acceptor fluorescence excitation (or absorption) spectrum ([http://www.nature.com/nprot/journal/v8/n2/full/nprot.2012.147.html Broussard et al., 2013]), as described in Fig.2.
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In biochemistry and cell biology fluorescent dyes, which interact via FRET, are applied as "optical nano metering rules", because the intensity of the transfer is dependent on the spacing between donor and acceptor. FRET can be observed over distances of up to 10&nbsp;nm. This way, protein-protein interactions and conformational changes of a variety of tagged biomolecules can be observed. For an efficient FRET to occur, there must be a substantial overlap between the donor fluorescence emission spectrum and the acceptor fluorescence excitation (or absorption) spectrum ([http://www.nature.com/nprot/journal/v8/n2/full/nprot.2012.147.html Broussard et al., 2013]), as described in the figure below.
{{Team:Aachen/Figure|Aachen_14-10-07_Jablonski_Diagram_and_Absorption_Spectra_iNB.png|align=center|title=FRET betweeen donor and acceptor|subtitle=On the left: Jablonski diagram showing the transfer of energy between donor and acceptor; on the right: For successful FRET, the emission spectrum of of the donor has to overlap with the absorption spectrum of the acceptor.|width=900px}}
{{Team:Aachen/Figure|Aachen_14-10-07_Jablonski_Diagram_and_Absorption_Spectra_iNB.png|align=center|title=FRET betweeen donor and acceptor|subtitle=On the left: Jablonski diagram showing the transfer of energy between donor and acceptor; on the right: For successful FRET, the emission spectrum of of the donor has to overlap with the absorption spectrum of the acceptor.|width=900px}}
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However, '''fluorescence is not an essential requirement for FRET'''. This type of energy transfer can also be observed between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence, and fit acceptors. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching instead. Thus, this kind of acceptors are also referred to as '''dark quenchers'''. In our project, we use a FRET system with a dark quencher, namely our '''REACh construct'''.
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However, '''fluorescence is not an essential requirement for FRET'''. This type of energy transfer can also be observed between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching instead. Thus, this kind of acceptors are also referred to as '''dark quenchers'''. In our project, we use a FRET system with a dark quencher, namely our '''REACh construct'''.
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In 2006, [http://www.pnas.org/content/103/11/4089.full Ganesan et al.] were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called '''REACh (for Resonance Energy-Accepting Chromoprotein)'''. YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption - as illustrated in Fig. 2 - but have separated emission spectra to allow their selective imaging.
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In 2006, [http://www.pnas.org/content/103/11/4089.full Ganesan et al.] were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called '''REACh (Resonance Energy-Accepting Chromoprotein)'''. YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption - as illustrated in previous section - but have separated emission spectra to allow their selective imaging.
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To optimize the spectral overlap of this FRET pair, the group obtained '''a genetically modified YFP acceptor'''. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98%, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors, and named them REACh1 and REACh2, respectively. '''Both REACh1 and REACh2 act as dark quenchers of GFP'''.
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To optimize the spectral overlap of this FRET pair, the group obtained '''a genetically modified YFP acceptor'''. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98&nbsp;%, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors, and named them REACh1 and REACh2, respectively. '''Both REACh1 and REACh2 act as dark quenchers of GFP'''.
 
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<span class="anchor" id="gfp-reach"></span>
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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.
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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 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 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] promoter''' that controls the expression of the TEV protease, which we made 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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The resulting fusion proteins were labelled as [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP fused with REACH1) and [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP fused with REACh 2) and the linker between the proteins is labelled as [http://parts.igem.org/Part:BBa_K1319013 K1319016].
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The resulting fusion proteins can be expressed in [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP fused with REACh1) and [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP fused with REACh2). The linker between the proteins containing a TEV protease cleavage site is labelled as [http://parts.igem.org/Part:BBa_K1319016 K1319016].
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Since we did not have enough time to build the complete system we tested our FRET reporter with an IPTG inducible TEV protease ([http://parts.igem.org/Part:BBa_K1319008 K1319008]). In this way we were able to establish a proof of concept of our reporter system including proper expression of the TEV protease as well as functionality of our GFP-REACh construct.
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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 promoter.|width=900px}}
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<span class="anchor" id="tevprotease"></span>
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To cut the GFP_REACh fusion protein off, we chose '''Tobacco Etch Virus (TEV) protease''', a highly sequence-specific cysteine protease, that is frequently used for the controlled cleavage of fusion proteins ''in vitro'' and ''in vivo''. The native protease also contains an internal self-cleavage site. This site is slowly cleaved to inactivate the enzyme. The physiological reason for the self-cleavage is unknown, however, undesired for our use. Therefore, our team uses a variant of the native TEV protease that contains the mutation S219V which results in an alteration of the cleavage site so that self-inactivation is diminished.
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To cleave the GFP-REACh fusion protein, we chose '''Tobacco Etch Virus (TEV) protease''', a highly sequence-specific cysteine protease, that is frequently used for the controlled cleavage of fusion proteins ''in vitro'' and ''in vivo''. The native protease also contains an internal self-cleavage site. This site is slowly cleaved to inactivate the enzyme. The physiological reason for the self-cleavage is unknown, however, undesired for our use. Therefore, our team uses a variant of the native TEV protease containing the mutation S219V which results in an alteration of the cleavage site so that self-inactivation is diminished.
-
{{Team:Aachen/Figure|Aachen_TEV_Protease_Model.png|title=TEV protease with a bound peptide|subtitle=This picture shows the TEV protease with a peptide chain bound in the binding pocket ready to be cleaved. The bound peptide chain has the recognition sequence inside the binding pocket. It is randered with POV-Ray.|width=800px}}
+
{{Team:Aachen/Figure|Aachen_TEV_Protease_Model.png|title=TEV protease with a bound peptide|subtitle=This picture shows the TEV protease with a peptide chain bound in the binding pocket ready to be cleaved. The recognition site of the bound peptide chain is located inside the binding pocket of the TEV protease. It was rendered with POV-Ray.|width=800px}}
 +
 
 +
Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the team Aachen introduces the protease with anti-self cleavage mutation S219V and codon optimized for ''E. coli'' [http://parts.igem.org/Part:BBa_K1319004 '''to the Parts Registry this year.''']
-
Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the Aachen team introduces a protease with anti-self cleavage mutation S219V and codon optimized for ''E. coli'' [http://parts.igem.org/Part:BBa_K1319004 '''to the Parts Registry this year.''']
 
{{Team:Aachen/BlockSeparator}}
{{Team:Aachen/BlockSeparator}}
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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>   
-
==Characterization of GFP-REACh1 and GFP-REACh 2 together with an IPTG inducible TEV protease==
+
===Characterization of GFP-REACh1 and GFP-REACh2 fusion proteins in combination 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 REACh1 and REACh2 dark quenchers was performed by introducing both simultaneously into ''E.&nbsp;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.  
-
As a positive control [http://parts.igem.org/Part:BBa_I20260 I20260] was used. I20260 contains the same promoter ([http://parts.igem.org/Part:BBa_J23101 J23101]] and RBS ([http://parts.igem.org/Part:BBa_B0032 B0032]) as well as the same fluorescence protein (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 we used [http://parts.igem.org/Part:BBa_B0015 B0015], a coding sequence of a Terminator which shouldn't show any sign of fluorescence.
+
[http://parts.igem.org/Part:BBa_I20260 I20260] was used as a positive control because I20260 contains the same promoter ([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 better evaluate the fluorescence we adjusted the fluorescence reading with the observed OD to achieve a fluorecence reading independent of the amount of cells present but rather representative of the amount of fluorescence per cell. With this the fluorescence of a constitutive expression, no expression and both of the REACh + TEV constructs induced and not induced are observed. 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.  
-
{{Team:Aachen/Figure|Aachen 16-10-14 Graph2iFG.PNG|title=Comparison of K1319013 + K1319008, K1319014 + K1319008, I20260 (positive control) and B0015 (negative control)|subtitle=Fluorescence was normalized by dividing by the optical density.|width=700px}}
+
To better evaluate the fluorescence, the observed Optical desity (OD) was taken into account in order to achieve a fluorescence measurement independent of the amount of cells present. This way, the measurement represents the amount of fluorescence per cell only.  
-
The negative control B0015 does not exhibit any significant fluuorescence as expected. The positive control I20260 shows a steady increase in fluorescence for the first X hours. After that the fluorescence stays constant due to the end of the exponential growth phase and the cells becoming stationary and not producing any more GFP. The production is also indpendent od the induction with IPTG as it is expected.  
+
{{Team:Aachen/Figure|Aachen 16-10-14 Graph2iFG.PNG|title=Comparison of K1319013 + K1319008, K1319014 + K1319008, I20260 (positive control) and B0015 (negative control)|subtitle=Both double plasmid construct exhibit a clear fluorescence signal when induced.|width=700px}}
-
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. It is also a partly due to an imperfect dark quenching of GFP by the REACh 1 and REACh 2 proteins.
+
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 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.  
+
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 promoter.  
-
===Summary===
+
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 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 fluorescence results of K1319014 + K1319008 was used to fit our [https://2014.igem.org/Team:Aachen/Project/Model model] to experimental data.
-
==Comparing the kinetic of the GFP-REACh fusion proteins with a standard lacI inducible GFP epression==
+
====Summary====
-
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.
+
The double plasmid systems of K1319013 + K1319008 and K1319014 + K1319008 demonstrate the quenching ability of the REACh1 and REACh2 proteins as well as the funcionality of the TEV protease.
 +
The increase in fluorescence after induction with IPTG is based on a functional expression of the TEV protease which proceeds to cut the linker of the fusion protein already produced destroying the FRET system between GFP and its quencher and resulting in a strong fluorescence signal. Combined, this characterization is a '''validation of the functionality of the REACh1 protein ([http://parts.igem.org/Part:BBa_K1319001 K1319001]), the REACh2 protein ([http://parts.igem.org/Part:BBa_K1319002 K1319002]) and the TEV protease ([http://parts.igem.org/Part:BBa_K1319004 K1319004])'''.
-
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.
+
===Determining the Quenching ability of REACh1 and REACh2===
-
{{Team:Aachen/Figure|Aachen_16-10-14_GraphQuotient_iFG.PNG|Comparison of K1319013 + K1319008, K1319014 + K1319008 and K731520|subtitle=Fluorescence was normalized by dividing by the optical density. The fluorescence of induced cells was additionally divided by the fluorescence of not induced cells.|width=700px}}
+
In order to further evaluate the quenching ability of the REACh1 and REACh2 constructs in the fusion proteins produced by K1319013 and K1319014, they were expressed alone without an IPTG inducible TEV protease. This eliminated the effect of a potential leakiness of the non induced promoter to reliably assess the quenching ability of the REACh1 and REACh2 proteins.  
 +
{{Team:Aachen/Figure|Aachen_K1319001_and_K1319002.PNG|title=Comparison of K1319013 and K1319014 with I20260 and B0015|subtitle=K1319013 and K1319014 show a severely reduced fluorescence compared to the positive control I20260.|width=700px}}
-
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.
+
In the previous experiment it was established that the fusion proteins K1319013 and K1319014 are expressed funtionally. K1319014 reached the same level of fluorescence as the positive control after being cut by the TEV protease. Therefore the reduced fluorescence in this experiment is completely attributable to the quenching of REACh1. The quenching reduces the fluorescence of GFP by a factor ~ 25 which means a '''quenching efficiency of ~ 96%!'''.  
-
===summary===
+
K139013 was not able to reach the same fluorescence level as the positive control in the previous experiment. Therefore a worse rate of functional expression of K1319013 compared to K1319014 is assumed. incorporating this, the difference in fluorescence between induced and not induced is still a factor of ~ 30 resulting in a  '''quenching efficiency of ~ 97%'''.
-
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.
+
====Summary ====
-
==Characterizing the GFP-REACh constructs in [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor sensor chips]==
+
The fusion proteins of GFP combined with REACh1 and REACh2 are not only fully functional but exhibit a great quenching efficiency of ~ 97% for REACh1 and ~ 96% for REACh2.Even though REACh1s quenching ability seems to be slighty superior, the expression level of the K1319014 fusion protein including REACh2 is nearly twice as high as the fusion protein K1319013 containing REACh1 and therefore shows a stronger fluorescence.  Ganesan et al. (2006) reported a reduction on emission of GFP of 82% for REACh1 and 98% for REACh2 but with a different Linker between the proteins and on a different vector backbone.
-
'''''Waiting for the Platereader data to be analyzed'''''
+
===Comparing fluorescence kinetics of the GFP-REACh fusion proteins with a Standard lacI-inducible GFP Expression===
 +
 
 +
To assess the kinetics of the fusion proteins K1319013 (GFP-REACh1) and K1319014 (GFP-REACh2), the double plasmid systems 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], a BioBrick made by the iGEM Team TRENTO in 2012. This tested the hypothesis of achieving a faster fluorescence response with the GFP-REACh fusion proteins compared to a standard expression.
 +
 
 +
K731520 and the double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 were cultivated in ''E. coli'' BL21(DE3), and fluorescence and OD was measured.The fluorescence was adjusted for the OD to show a relative fluorescence on a per cell basis. The difference between the induced and non-induced state, the fluorescence quotient, serves as a better indicator for a system used as a sensor because the difference between an ''on'' and ''off'' state is more important for a clear and unmistakable signal than the overall fluorescence. Hence, the OD-adjusted fluorescence quotient for both double plasmid constructs and K731520 was obtained and plotted in the following graph.
 +
 
 +
{{Team:Aachen/Figure|Aachen_16-10-14_GraphQuotient_iFG.PNG|Comparison of K1319013 + K1319008, K1319014 + K1319008 and K731520|subtitle=Fluorescence was normalized to the cell growth. The fluorescence of induced cells was additionally divided by the fluorescence of non-induced cells to obtain the fluorescence quotient.|width=700px}}
 +
 
 +
The graph clearly shows the faster response 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 between induced and non-induced state as well as at a faster rate. This proves the hypothesis made earlier about the kinetics of the GFP-REACh fusion protein combined with the TEV protease.
 +
 
 +
====Summary====
 +
 
 +
The kinetics of the fusion protein combined with the TEV protease exhibits the exact characteristics as predicted. The response is clearly faster than normal expression by accumulating a reservoir of fusion proteins which are not fluorescing due to the dark quencher attached to them. This reservoir is then activated by the induction of the TEV protease expression. Production of the protease results in the cleavage of the fusion protein, releasing GFP from the dark quencher and disturbing the interaction between the FRET pair. This results in the observed faster fluorescence reaction due to the amplificating effect of the TEV protease in which every one TEV protease can account for many fluorescence proteins being activated.
 +
 
 +
===Characterizing the GFP-REACh Constructs in Sensor Chips===
 +
To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2&nbsp;µL IPTG with a concentration of 100&nbsp;mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496&nbsp;±&nbsp;9&nbsp;nm, emission&nbsp;516&nbsp;±&nbsp;9&nbsp;nm) roughly every 10&nbsp;min in the plate reader. The results were plotted in the heatmap shown here.
 +
<center>
 +
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|500px}}};padding:10px 10px 0px 0px;">
 +
{|
 +
|<html> <img src="https://static.igem.org/mediawiki/2014/6/6d/Aachen_K1319014%2B8_and_K1319013%2B8_pixelig_minus_bg.gif" width="500px"></html>
 +
|-
 +
|'''{{{title|K1319014 + K1319008 and K1319013 + K1319008 non-induced (top) and induced (bottom) in sensor cells }}}'''<br />{{{subtitle|The induced double plasmid systems K1319013 + K1319008 and K1319014&nbsp;+&nbsp;K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG. }}}
 +
|}
 +
</div>
 +
</center>
 +
 
 +
The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This shows that the constructs also work  as intended in the sensor chips: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease.
 +
 
 +
===Comparing the kinetic of the double plasmid systems K1319013 + K1319008 and K1319014 + K1319008 with standard GFP expression===
 +
 
 +
<center>
 +
<div class="figure" style="float:{{{align|center}}}; margin: 0px 10px 10px 40px; border:{{{border|0px solid #aaa}}};width:{{{width|480px}}};padding:10px 10px 0px 0px;">
 +
{|
 +
|<html> <img src="https://static.igem.org/mediawiki/2014/1/1a/Aachen_K13%2B8%2CK14%2B8%2CK731_slower_reduced.gif" width="480px"></html>
 +
|-
 +
|'''{{{title|K1319013 + K1319008, K1319014 + K1319008 and K731520 in an non-induced (top) and induced (bottom) chip}}}'''<br />{{{subtitle|Comparing the factor of fluorescence increase of induced (bottom) and not induced (top) sensor chips to the first frame of the measurement. The tested constructs are K1319013 + K1319008, K1319014 + K1319008 and K731520. After five hours, the fluorescence in our dual-plasmid REACh construct contaning K1319014 had increased by a factor of about 8, while K731520 fluorescence increased by only a factor of 2-3.}}}
 +
|}
 +
</div>
 +
</center>
 +
 
 +
The analysis of the different [https://2014.igem.org/Team:Aachen/Project/2D_Biosensor sensor chips] with the three different construct K1319013 + K1319008, K1319014 + K1319008 and K731520 demonstrates the same fluorescence response kinetic as in the shake flask experiments. The double plasmid systems exhibits a faster and stronger fluorescence response compared to a standard GFP expression in K731520. The build up pool of fusion proteins allows for a faster, stronger fluorescence response when the induced TEV protease cleaves the fusion proteins and releases GFP from its dark quencher.
{{Team:Aachen/BlockSeparator}}
{{Team:Aachen/BlockSeparator}}
-
=Outlook=
+
[[File:Aachen_14-10-16_Outlook_Cellocks_iNB.png|right|150px]]
-
The system of the GFP-REACh fusion proteins with an inducible TEV protease has been establsihed and shows clearly the desired results of being faster than normal expression. The next step will be to engineer the TEV protease to be inducible by the HSL instead of IPTG and then to incorporate both the inducible TEV protease and the fusion protein on one plasmid backbone. This would also allow us to choose a high copy plasmid for both inserts, instead of a high copy plasmid for the TEV protease and a low to mid copy plasmid for the fusion protein which should yield an overall higher fluorescence readout.
+
== Outlook ==
 +
<span class="anchor" id="reachoutlook"></span>
-
Afterwards we would then like to characterize this construct the same way we have done with the double plasmid system and explore option for other fluorescence proteins than GFP to incorporate into our system with different quenchers to be able to have multiple fluorescence responses readable at the same time while being fast than the normal expression.  
+
The system of the GFP-REACh fusion proteins with an inducible TEV protease has been established and shows the desired results of being faster than standard expression. The next step will be to devise an expression of the TEV protease inducible by HSL instead of IPTG and then to incorporate both the HSL inducible TEV protease and the fusion protein into one plasmid backbone. This would also allow us to choose a high copy plasmid for both inserts, instead of a high copy plasmid for the TEV protease and a low to mid copy plasmid for the fusion protein which should yield an overall higher fluorescence readout.
 +
 
 +
Subsequently the combined construct will be characterizes the same way as the double plasmid system and other options regarding different fluorescence proteins with different quenchers will be considered to be able to have multiple fluorescence responses readable at the same time while still being faster than normal expression.
 +
 
 +
Also finding and testing different promoters which can be used to express the TEV protease is planned to be able to detect not only ''Pseudomonas aeruginosa'' but also other pathogens. Also the technology can be expanded to other molecules enabling other research areas to profit from the faster fluorescence response.
-
Also finding and testing different promoters to induce the TEV protease is planned to be able to detect not only ''Pseudomonas aeruginosa'' but also other pathogens or other relevant molecules in general so that we can establish a concept for faster recognition for a variety of uses.
 
{{Team:Aachen/BlockSeparator}}
{{Team:Aachen/BlockSeparator}}
= References =
= References =
 +
* Sundar Ganesan, Simon M. Ameer-Beg, Tony T. C. Ng, Borivoj Vojnovic and Fred S. Wouters. "A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP" Proceedings of the National Academy of Science of the United States of America | March 14,2006 | vol. 103 | no. 11 | 4089 - 4094
 +
* Broussard, Joshua A, Benjamin Rappaz, Donna J Webb, and Claire M Brown. "Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt." Nature protocols 8.2 (2013): 265-281. doi:10.1038/nprot.2012.147.  
* Broussard, Joshua A, Benjamin Rappaz, Donna J Webb, and Claire M Brown. "Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt." Nature protocols 8.2 (2013): 265-281. doi:10.1038/nprot.2012.147.  
-
* Ganesan, Sundar, Simon M. Ameer-beg, Tony T. C. Ng, Borivoj Vojnovic, and Fred S. Wouters. " A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP." Proceedings of the National Academy of Sciences of the United States of America 103.11 (2006): 4089–4094. doi: 10.1073/pnas.0509922103  
+
* Broussard, J. A., Rappaz, B., Webb, D. J., & Brown, C. M. (2013). Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nature protocols, 8(2), 265-281. doi: 10.1073/pnas.0509922103  
'''SWISS-MODEL'''
'''SWISS-MODEL'''
-
* Marco Biasini, Stefan Bienert, Andrew Waterhouse, Konstantin Arnold, Gabriel Studer, Tobias Schmidt, Florian Kiefer, Tiziano Gallo Cassarino, Martino Bertoni, Lorenza Bordoli, Torsten Schwede. (2014). SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research; doi: 10.1093/nar/gku340.
+
* Arnold, K., Bordoli, L., Kopp, J., & Schwede, T. (2005). The SWISS-MODEL Workspace: A Web-based Environment For Protein Structure Homology Modelling. Bioinformatics, 22(2), 195-201.
-
* Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201.  
+
 
-
* Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research. 37, D387-D392.  
+
* Biasini, M., Bienert, S., Schwede, T., Waterhouse, A., Arnold, K., Studer, G., et al. (2014). Nucleic Acids Research. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. doi: 10.1093/nar/gku340.
-
* Guex, N., Peitsch, M.C., Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis, 30(S1), S162-S173.  
+
 
 +
* Guex, N., Peitsch, M. C., & Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis, 30(S1), S162-S173.
 +
 
 +
* Kiefer, F., Arnold, K., Kunzli, M., Bordoli, L., & Schwede, T. (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research, 37(Database), D387-D392.
'''UCSF Chimera'''
'''UCSF Chimera'''
-
* Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera--a visualization system for exploratory research and analysis. JComput Chem. 2004 Oct;25(13):1605-12. PubMed PMID: 15264254.
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* Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., et al. (2004). UCSF Chimera?A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25(13), 1605-1612. PubMed PMID: 15264254.
'''POV-Ray'''
'''POV-Ray'''
* Persistence of Vision Pty. Ltd. (2004) Persistence of Vision Raytracer (Version 3.7) [Computer software]. Retrieved from http://www.povray.org/download/
* Persistence of Vision Pty. Ltd. (2004) Persistence of Vision Raytracer (Version 3.7) [Computer software]. Retrieved from http://www.povray.org/download/
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{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Latest revision as of 03:47, 18 October 2014

The modified REACh Construct

On this page, we present our biosensor on a molecular level. Here you can explore the different parts of our genetic device:


Aachen 14-10-13 GFP iNB.png

A Fluorescence Answer Faster than Expression

Biosensor systems are usually based on a reporter gene under the control of a promoter directly induced by the biomolecule of interest. In the case of our 2D biosensor for Pseudomonas aeruginosa, the expression of our fluorescing reporter protein is directly induced by the activity of the bacterium's quorum sensing molecules. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we aimed to develop a system that provides a fluorescent answer faster than just expressing the fluorescent protein GFP.

Aachen Traditional biosensor.png
Schematic model of a traditional biosensor
In this model, the expression of GFP is directly controlled by a promoter whose activator binds to a molecule secreted by the pathogen.

As an alternative to the traditional approach, we constitutively express our reporter gene in a quenched form. By fusing GFP to REACh, a mutated variant of EYFP, its original fluorescence is thoroughly suppressed. Thereby, a huge stock of temporary inactivated fluorescence proteins is already available prior to first contact with the pathogen. As a conseqence to the uptake of homoserine lactones of P. aeruginosa and binding to the LasI promoter in front of the protease gene, these autoinducers activate the expression of the TEV protease. This way, our biosensor provides a fast increase in fluorescence intensity by cleavage of GFP-REACh-constructs.

Aachen REACh approach.png
Schematic model of our novel biosensor
Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elicit a fluorescence response.

Advantages of the novel biosensor:

  • When P. aeruginosa is detected by our cells, the reporter protein has already been expressed and only waits to be activated. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. Therefore, our sensor cells provide an exceptionally early response.
  • While a certain concentration of homoserine lactone will produce the same number of gene read-outs and the TEV protease is about the same size as GFP (~27 kDa), one TEV protease can cleave many GFP-REACh constructs per time than fluorescent protein molecules could be expressed and go through fluorescence maturation. Through this additional reaction, we introduce an amplification step into our system. Hypothetically, the TEV protease will enable the production of a much stronger signal in a given time interval.

In parallel, we worked on a model of the respective biosensor system, in order to confirm our hypothesis of a faster and stronger fluorescence response than a conventional biosensor would povide.


Aachen 14-10-13 FRET Arrows iNB.png

The FRET (Förster Resonance Energy Transfer) System

Förster resonance energy transfer (FRET), sometimes also called fluorescence resonance energy transfer, is a physical process of energy transfer. In FRET, the energy of a donor chromophore, whose electrons are in an excited state, is passed to a second chromophore, the acceptor. The energy is transferred without radiation and is therefore not exchanged via emission and absorption of photons. The acceptor then releases the energy received from the donor, for example, as light of a longer wavelength.

In biochemistry and cell biology fluorescent dyes, which interact via FRET, are applied as "optical nano metering rules", because the intensity of the transfer is dependent on the spacing between donor and acceptor. FRET can be observed over distances of up to 10 nm. This way, protein-protein interactions and conformational changes of a variety of tagged biomolecules can be observed. For an efficient FRET to occur, there must be a substantial overlap between the donor fluorescence emission spectrum and the acceptor fluorescence excitation (or absorption) spectrum ([http://www.nature.com/nprot/journal/v8/n2/full/nprot.2012.147.html Broussard et al., 2013]), as described in the figure below.

Aachen 14-10-07 Jablonski Diagram and Absorption Spectra iNB.png
FRET betweeen donor and acceptor
On the left: Jablonski diagram showing the transfer of energy between donor and acceptor; on the right: For successful FRET, the emission spectrum of of the donor has to overlap with the absorption spectrum of the acceptor.

However, fluorescence is not an essential requirement for FRET. This type of energy transfer can also be observed between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching instead. Thus, this kind of acceptors are also referred to as dark quenchers. In our project, we use a FRET system with a dark quencher, namely our REACh construct.


Aachen 14-10-13 REACh iNB.png

REACh Proteins - Dark Quenchers of GFP

256px
Homology model of REACh.
This homology model was created with SWISS-MODEL. We used Chimera to prepare and export a scene that was then rendered into an animation with POV-Ray.

In 2006, [http://www.pnas.org/content/103/11/4089.full Ganesan et al.] were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called REACh (Resonance Energy-Accepting Chromoprotein). YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption - as illustrated in previous section - but have separated emission spectra to allow their selective imaging.

To optimize the spectral overlap of this FRET pair, the group obtained a genetically modified YFP acceptor. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98%, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors, and named them REACh1 and REACh2, respectively. Both REACh1 and REACh2 act as dark quenchers of GFP.


Aachen 14-10-13 Fusion Protein iNB.png

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 [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 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 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] promoter that controls the expression of the TEV protease, which we made 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.

Aachen 14-10-08 REACh approach with BioBricks iNB.png
Composition of our biosensor
For our biosensor, we use a mix of already available and self-constructed BioBricks.

The resulting fusion proteins can be expressed in [http://parts.igem.org/Part:BBa_K1319013 K1319013] (GFP fused with REACh1) and [http://parts.igem.org/Part:BBa_K1319014 K1319014] (GFP fused with REACh2). The linker between the proteins containing a TEV protease cleavage site is labelled as [http://parts.igem.org/Part:BBa_K1319016 K1319016].

Since we did not have enough time to build the complete system we tested our FRET reporter with an IPTG inducible TEV protease ([http://parts.igem.org/Part:BBa_K1319008 K1319008]). In this way we were able to establish a proof of concept of our reporter system including proper expression of the TEV protease as well as functionality of our GFP-REACh construct.


Aachen 14-10-17 IPTG REACh iFG.png
Composition of our biosensor for IPTG
To test the funtionality of our sensor concept, we expressed the TEV protease with a T7 promoter.


Aachen 14-10-13 TEV Protease iNB.png

Cutting the Fusion Protein with the TEV Protease

To cleave the GFP-REACh fusion protein, we chose Tobacco Etch Virus (TEV) protease, a highly sequence-specific cysteine protease, that is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo. The native protease also contains an internal self-cleavage site. This site is slowly cleaved to inactivate the enzyme. The physiological reason for the self-cleavage is unknown, however, undesired for our use. Therefore, our team uses a variant of the native TEV protease containing the mutation S219V which results in an alteration of the cleavage site so that self-inactivation is diminished.

Aachen TEV Protease Model.png
TEV protease with a bound peptide
This picture shows the TEV protease with a peptide chain bound in the binding pocket ready to be cleaved. The recognition site of the bound peptide chain is located inside the binding pocket of the TEV protease. It was rendered with POV-Ray.

Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the team Aachen introduces the protease with anti-self cleavage mutation S219V and codon optimized for E. coli [http://parts.igem.org/Part:BBa_K1319004 to the Parts Registry this year.]


Aachen 14-10-15 Medal Cellocks iNB.png

Achievements

Characterization of GFP-REACh1 and GFP-REACh2 fusion proteins in combination with an IPTG-inducible TEV Protease

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 promoter ([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 better evaluate the fluorescence, the observed Optical desity (OD) was taken into account in order to achieve a fluorescence measurement independent of the amount of cells present. This way, the measurement represents the amount of fluorescence per cell only.

Aachen 16-10-14 Graph2iFG.PNG
Comparison of K1319013 + K1319008, K1319014 + K1319008, I20260 (positive control) and B0015 (negative control)
Both double plasmid construct exhibit a clear fluorescence signal when induced.

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 promoter.

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 fluorescence results of K1319014 + K1319008 was used to fit our model to experimental data.

Summary

The double plasmid systems of K1319013 + K1319008 and K1319014 + K1319008 demonstrate the quenching ability of the REACh1 and REACh2 proteins as well as the funcionality of the TEV protease. The increase in fluorescence after induction with IPTG is based on a functional expression of the TEV protease which proceeds to cut the linker of the fusion protein already produced destroying the FRET system between GFP and its quencher and resulting in a strong fluorescence signal. Combined, this characterization is a validation of the functionality of the REACh1 protein ([http://parts.igem.org/Part:BBa_K1319001 K1319001]), the REACh2 protein ([http://parts.igem.org/Part:BBa_K1319002 K1319002]) and the TEV protease ([http://parts.igem.org/Part:BBa_K1319004 K1319004]).

Determining the Quenching ability of REACh1 and REACh2

In order to further evaluate the quenching ability of the REACh1 and REACh2 constructs in the fusion proteins produced by K1319013 and K1319014, they were expressed alone without an IPTG inducible TEV protease. This eliminated the effect of a potential leakiness of the non induced promoter to reliably assess the quenching ability of the REACh1 and REACh2 proteins.

Aachen K1319001 and K1319002.PNG
Comparison of K1319013 and K1319014 with I20260 and B0015
K1319013 and K1319014 show a severely reduced fluorescence compared to the positive control I20260.

In the previous experiment it was established that the fusion proteins K1319013 and K1319014 are expressed funtionally. K1319014 reached the same level of fluorescence as the positive control after being cut by the TEV protease. Therefore the reduced fluorescence in this experiment is completely attributable to the quenching of REACh1. The quenching reduces the fluorescence of GFP by a factor ~ 25 which means a quenching efficiency of ~ 96%!.

K139013 was not able to reach the same fluorescence level as the positive control in the previous experiment. Therefore a worse rate of functional expression of K1319013 compared to K1319014 is assumed. incorporating this, the difference in fluorescence between induced and not induced is still a factor of ~ 30 resulting in a quenching efficiency of ~ 97%.

Summary

The fusion proteins of GFP combined with REACh1 and REACh2 are not only fully functional but exhibit a great quenching efficiency of ~ 97% for REACh1 and ~ 96% for REACh2.Even though REACh1s quenching ability seems to be slighty superior, the expression level of the K1319014 fusion protein including REACh2 is nearly twice as high as the fusion protein K1319013 containing REACh1 and therefore shows a stronger fluorescence. Ganesan et al. (2006) reported a reduction on emission of GFP of 82% for REACh1 and 98% for REACh2 but with a different Linker between the proteins and on a different vector backbone.

Comparing fluorescence kinetics of the GFP-REACh fusion proteins with a Standard lacI-inducible GFP Expression

To assess the kinetics of the fusion proteins K1319013 (GFP-REACh1) and K1319014 (GFP-REACh2), the double plasmid systems 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], a BioBrick made by the iGEM Team TRENTO in 2012. This tested the hypothesis of achieving a faster fluorescence response with the GFP-REACh fusion proteins compared to a standard expression.

K731520 and the double plasmid constructs K1319013 + K1319008 and K1319014 + K1319008 were cultivated in E. coli BL21(DE3), and fluorescence and OD was measured.The fluorescence was adjusted for the OD to show a relative fluorescence on a per cell basis. The difference between the induced and non-induced state, the fluorescence quotient, serves as a better indicator for a system used as a sensor because the difference between an on and off state is more important for a clear and unmistakable signal than the overall fluorescence. Hence, the OD-adjusted fluorescence quotient for both double plasmid constructs and K731520 was obtained and plotted in the following graph.

Aachen 16-10-14 GraphQuotient iFG.PNG

Fluorescence was normalized to the cell growth. The fluorescence of induced cells was additionally divided by the fluorescence of non-induced cells to obtain the fluorescence quotient.

The graph clearly shows the faster response 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 between induced and non-induced state as well as at a faster rate. This proves the hypothesis made earlier about the kinetics of the GFP-REACh fusion protein combined with the TEV protease.

Summary

The kinetics of the fusion protein combined with the TEV protease exhibits the exact characteristics as predicted. The response is clearly faster than normal expression by accumulating a reservoir of fusion proteins which are not fluorescing due to the dark quencher attached to them. This reservoir is then activated by the induction of the TEV protease expression. Production of the protease results in the cleavage of the fusion protein, releasing GFP from the dark quencher and disturbing the interaction between the FRET pair. This results in the observed faster fluorescence reaction due to the amplificating effect of the TEV protease in which every one TEV protease can account for many fluorescence proteins being activated.

Characterizing the GFP-REACh Constructs in Sensor Chips

To further characterize the REACh construct, they were introduced into the sensor cells which were then induced with 2 µL IPTG with a concentration of 100 mM. Subsequently, we took fluorescence measurement read-outs (GFP, excitation 496 ± 9 nm, emission 516 ± 9 nm) roughly every 10 min in the plate reader. The results were plotted in the heatmap shown here.

K1319014 + K1319008 and K1319013 + K1319008 non-induced (top) and induced (bottom) in sensor cells
The induced double plasmid systems K1319013 + K1319008 and K1319014 + K1319008 exhibit a clear fluorescence response in our sensor cells which in response to induction with 2 µl IPTG.

The heatmap shows an increase of fluorescence from blue (no fluorescence) to red (high fluorescence). It is clearly visible that the induced chips are exhibiting a significantly higher fluorescence than the non-induced chips. This shows that the constructs also work as intended in the sensor chips: The TEV protease cuts the linker so that the fusion protein is separated into GFP and a dark quencher, disabling the quenching. GFP has a clear fluorescence emission after the fusion protein has been successfully cut into two pieces by the TEV protease.

Comparing the kinetic of the double plasmid systems K1319013 + K1319008 and K1319014 + K1319008 with standard GFP expression

K1319013 + K1319008, K1319014 + K1319008 and K731520 in an non-induced (top) and induced (bottom) chip
Comparing the factor of fluorescence increase of induced (bottom) and not induced (top) sensor chips to the first frame of the measurement. The tested constructs are K1319013 + K1319008, K1319014 + K1319008 and K731520. After five hours, the fluorescence in our dual-plasmid REACh construct contaning K1319014 had increased by a factor of about 8, while K731520 fluorescence increased by only a factor of 2-3.

The analysis of the different sensor chips with the three different construct K1319013 + K1319008, K1319014 + K1319008 and K731520 demonstrates the same fluorescence response kinetic as in the shake flask experiments. The double plasmid systems exhibits a faster and stronger fluorescence response compared to a standard GFP expression in K731520. The build up pool of fusion proteins allows for a faster, stronger fluorescence response when the induced TEV protease cleaves the fusion proteins and releases GFP from its dark quencher.


Aachen 14-10-16 Outlook Cellocks iNB.png

Outlook

The system of the GFP-REACh fusion proteins with an inducible TEV protease has been established and shows the desired results of being faster than standard expression. The next step will be to devise an expression of the TEV protease inducible by HSL instead of IPTG and then to incorporate both the HSL inducible TEV protease and the fusion protein into one plasmid backbone. This would also allow us to choose a high copy plasmid for both inserts, instead of a high copy plasmid for the TEV protease and a low to mid copy plasmid for the fusion protein which should yield an overall higher fluorescence readout.

Subsequently the combined construct will be characterizes the same way as the double plasmid system and other options regarding different fluorescence proteins with different quenchers will be considered to be able to have multiple fluorescence responses readable at the same time while still being faster than normal expression.

Also finding and testing different promoters which can be used to express the TEV protease is planned to be able to detect not only Pseudomonas aeruginosa but also other pathogens. Also the technology can be expanded to other molecules enabling other research areas to profit from the faster fluorescence response.


References

  • Sundar Ganesan, Simon M. Ameer-Beg, Tony T. C. Ng, Borivoj Vojnovic and Fred S. Wouters. "A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP" Proceedings of the National Academy of Science of the United States of America | March 14,2006 | vol. 103 | no. 11 | 4089 - 4094
  • Broussard, Joshua A, Benjamin Rappaz, Donna J Webb, and Claire M Brown. "Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt." Nature protocols 8.2 (2013): 265-281. doi:10.1038/nprot.2012.147.
  • Broussard, J. A., Rappaz, B., Webb, D. J., & Brown, C. M. (2013). Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nature protocols, 8(2), 265-281. doi: 10.1073/pnas.0509922103

SWISS-MODEL

  • Arnold, K., Bordoli, L., Kopp, J., & Schwede, T. (2005). The SWISS-MODEL Workspace: A Web-based Environment For Protein Structure Homology Modelling. Bioinformatics, 22(2), 195-201.
  • Biasini, M., Bienert, S., Schwede, T., Waterhouse, A., Arnold, K., Studer, G., et al. (2014). Nucleic Acids Research. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. doi: 10.1093/nar/gku340.
  • Guex, N., Peitsch, M. C., & Schwede, T. (2009). Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis, 30(S1), S162-S173.
  • Kiefer, F., Arnold, K., Kunzli, M., Bordoli, L., & Schwede, T. (2009). The SWISS-MODEL Repository and associated resources. Nucleic Acids Research, 37(Database), D387-D392.

UCSF Chimera

  • Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., et al. (2004). UCSF Chimera?A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25(13), 1605-1612. PubMed PMID: 15264254.

POV-Ray

  • Persistence of Vision Pty. Ltd. (2004) Persistence of Vision Raytracer (Version 3.7) [Computer software]. Retrieved from http://www.povray.org/download/