http://2012.igem.org/wiki/index.php?title=Special:Contributions/Fabian_Froehlich&feed=atom&limit=50&target=Fabian_Froehlich&year=&month=2012.igem.org - User contributions [en]2024-03-28T13:43:05ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:57:14Z<p>Fabian Froehlich: /* LexA Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
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An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10 seconds and a pulse frequency of 1 pulse every 5 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the LexA binding site [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031] itself contains four additional TATA boxes which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]] Jayaram, M., Li, Y. Y., and Broach, J. R. (1983). The yeast plasmid 2mu circle encodes components required for its high copy propagation. ''Cell'', 34(1):95–104.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:46:52Z<p>Fabian Froehlich: /* Induction Setup */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10 seconds and a pulse frequency of 1 pulse every 5 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the promoter itself contains a TATA box which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]] Jayaram, M., Li, Y. Y., and Broach, J. R. (1983). The yeast plasmid 2mu circle encodes components required for its high copy propagation. ''Cell'', 34(1):95–104.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T01:46:09Z<p>Fabian Froehlich: /* Light-switchable promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to proof the production of limonene in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/OverviewTeam:TU Munich/Project/Overview2012-10-27T01:45:30Z<p>Fabian Froehlich: /* Light-switchable promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
<br />
= Overview =<br />
<hr/><br />
<br />
== Vision ==<br />
----<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
==Biosynthesis pathways==<br />
----<br />
<br />
<div class="bezel mfull"><br />
===Limonene===<br />
Limonene is a cyclic terpene and a major constituent of several citrus oils. D-limonene has been used as a component of flavorings and fragrances. It is formed from geranyl pyrophosphate by limonene synthase.<br />
<br />
We successfully demonstrated the production of the flavoring substance limonene by expressing limonene synthase in ''S. cerevisiae'', which naturally synthesizes the educt geranyl pyrophosphate.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_limonene.png|500px|thumb|right| Main results from our limonene subproject: reaction mechanism (A), constructed BioBricks (B) and proof of principle for the in vivo production of limonene]]<br />
<center>'''Experimental results:'''</center><br><br />
(+)-limonene synthase 1 (<partinfo>BBa_K801065</partinfo>) and (+)-limonene synthase 1 with yeast consensus sequence (<partinfo>BBa_K801060</partinfo>) were successfully cloned into our new yeast expression vector pTUM104. Expression of recombinant limonene synthase in ''Saccharomyces cerevisiae'' was proven by western blotting. Subsequently the protein was purified using SA-chromatography and size exclusion chromatography. The functionality of the enzyme was verified by ''in vivo'' and ''in vitro'' detection of limonene via GC-MS. <br />
<br />
Furthermore, we established gene constructs of the limonene synthase coding sequence with different yeast specific promoters and terminators (<partinfo>BBa_K801062</partinfo>, <partinfo>BBa_K801063</partinfo> and <partinfo>BBa_K801064</partinfo>). <br />
<br />
Last but not least, we have brewed iGEM's first SynBio beer containing limonene.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
We have achieved functional expression of ''Citrus limon'' limonene synthase and production of limonene in yeast. Furthermore, we have been able to proof the production of limonene in the beers we brewed. Further experiments regarding the concentrations of limonene produced will be carried out in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Limonene"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Thaumatin===<br />
Thaumatin is a natural protein which is synthesized by the katamfe plant (''Thaumatococcus daniellii''). It is said to be 2,000 to 100,000 times sweeter than sucrose on molar basis, but the sweetness builds up slow and lasts long. It has been approved as a sweetener by the European Union (E957).<br />
<br />
Our aim is to have ''S. cerevisiae'' secrete functional thaumatin by expressing preprothaumatin – a principle which has been proven by [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]].<br />
<hr><br />
[[file:TUM12_experiment_overwiew_Thaumati.png|500px|thumb|right| Main results from the Thaumatin subproject: Structure of Thaumatin (A), constructed BioBricks (B), profile of an ion exchange chromatography (IEC) used to detect our recombinant Thaumatin and a SDS-PAGE gel showing IEC elution fractions containing Thaumatin]]<br />
<center>'''Experimental results:'''</center><br><br />
The BioBrick for preprothaumatin [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080] as well as an expression cassette [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 K801080] were successfully cloned, expressed in yeast, purified using an ion exchange chromatography (see figure C) and detected in the SDS-PAGE. Therefore, the expression of thaumatin in yeast could be demonstrated and functionality of the BioBrick is confirmed.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
A proof of principle for the expression of thaumatin was achieved. Further goals are the increase of the expression of thaumatin and the investigation of the secretion.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Thaumatin"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Caffeine===<br />
Caffeine is a purine-alkaloid and its biosynthesis is known from coffee and tea plants. The molecule acts as a competitive antagonist of adenosine receptors and, therefore, increases indirectly neurotransmitter concentrations resulting in warding off drowsiness and restoring alertness. <br />
<br />
The idea is to perform a heterologous gene expression of the three enzymes 7-methylxanthosine synthase (CaXMT1), N-methyl nucleosidase (CaMXMT1) and caffeine synthase (CaDXMT1) required for caffeine biosynthesis in ''Saccharomyces cerevisiae''. <br />
<br />
<br />
<hr><br />
[[file:TUM12_Overviewcaffeine.png|500px|thumb|right| Figure showing a schematic overview of the reaction in (A), a western blot against the Strep-tag II for GFP (lane 1), [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801070 BBa_K801070]] (lane 2) and [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801071 BBa_K801071]] (lane 3) in (B) and finally the same western blot development for [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801072 BBa_K801072]] (C) LC/MS spectra of ''in vitro'' caffeine synthesis enzyme assay, showing produced theobromine]]<br />
<center>'''Experimental results:'''</center><br><br />
* Successful cloning of the three enzymes [http://partsregistry.org/Part:BBa_K801070 7-methylxanthosine synthase (CaXMT1)], [http://partsregistry.org/Part:BBa_K801071 theobromine synthase (CaMXMT1)] and the [http://partsregistry.org/Part:BBa_K801072 caffeine synthase (CaDXMT1)] into the shuttle vector pTUM104 and pSB1C3 each. <br />
* Successful assembly of the BioBricks to form expression cassettes consisting of promoter, gene and terminator: [http://partsregistry.org/Part:BBa_K801073 pTEF2-CaXMT1-tADH1], [http://partsregistry.org/Part:BBa_K801074 pTEF1-CaMXMT1-tADH1] and [http://partsregistry.org/Part:BBa_K801075 pTEF2-CaDXMT1-tADH1]) into pSB1C3.<br />
* Successful assembly of the expression cassettes of the three relevant enzymes forming a composite part of 6.4 kb capable of caffeine production in yeast ([http://partsregistry.org/Part:BBa_K801077 Caffeine Synthesis Pathway]) into pSB1C3.<br />
* Successful expression of CaXMT1, CaMXMT1 and CaDXMT1 in ''Saccharomyces cerevisiae'' INVSc1 in selective Sc minimal induction medium lacking uracil with 2 % galactose.<br />
<br />
<br />
<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
The homologue expression of the three required enzymes for caffeine synthesis in ''Saccharomyces cerevisiae'' INVSc1 transformed with pTUM102_CaXMT1, pTUM102_CaMXMT1 and pTUM102_CaDXMT1 was successful. Further testing of caffeine production using crude extracts from lysed yeast cells which had previously been transformed with our caffeine synthesis expression cartridge has been done and we were successful in producing theobromine, the immediate precursor of caffein, which we detected by the use of LC/MS with multiple reaction monitoring (MRM). <br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Caffeine"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Xanthohumol===<br />
Xanthohumol is known as a putative cancer chemopreventive agent due to its antioxidant activities [[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]]. Our goal is a heterologous gene expression of all enzymes required for xanthohumol biosynthesis in ''S. cerevisiae''.<br />
<br />
The pathway for the production of this plant secondary metabolite is composed of five steps, starting with the conversion of tyrosine and followed by four further enzymatic reactions.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_xanto2.png|500px|thumb|right| Main results from the Xanthohumol subproject: Successful reconstruction of the metabolic pathway for Xanthohumol]]<br />
<center>'''Experimental results:'''</center><br><br />
The whole biosynthetic pathway for the production of xanthohumol was converted into BioBricks. Except for APT each of the enzymes were cloned in two versions one having the proposed consensus sequence for more efficient expression in yeast chassis and another for usage of these BioBricks in other chassis. All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts:<br />
PAL (<partinfo>BBa_K801090</partinfo>, <partinfo>BBa_K801091</partinfo>), 4CL (<partinfo>BBa_K801092</partinfo>, <partinfo>BBa_K801093</partinfo>), CHS (<partinfo>BBa_K801094</partinfo>, <partinfo>BBa_K801095</partinfo>), APT (<partinfo>BBa_K801096</partinfo> and OMT (<partinfo>BBa_K801097</partinfo>, <partinfo>BBa_K801098</partinfo>). <br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
The construction of the xanthohumol pathway was achieved, whereas the expression and characterization might be an interesting task for iGEM teams in the future.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Xanthohumol"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Vector Design==<br />
----<br />
<div class="bezel mfull"><br />
===pTUM100===<br />
Designing an expression vector for yeast which is compatible to the iGEM cloning principles and standards was the main aim of this subproject. Based on the commercially available pYES2 vector we created vectors containing inducible and constitutive promoters in order to establish efficient possibilities to clone and express our enzymes.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_vector.png|500px|thumb|right| Explanations on the figure:<br />
<br />
Figure A shows the new multiple cloning site (MCS) containing the RFC 10/25 restriction sides and the DNA sequence coding for the ''Strep''-tag II.<br />
<br />
Figure B gives an overview of all important functional elements located on the vector backbone. Upstream to the new MCS lies a T7 promoter primer binding site allowing easy forward sequencing of integrated gene constructs using the standard T7 primer. The URA 3 gene is a prototrophy marker used for the selection of transfected cells.<br />
<br />
Figure C to E present the successfully designed BioBricks: pTUM100 simply contains the new MCS, the transcription terminator and further elements required for cloning and transfection. pTUM102 to pTUM104 contain in addition the constitutive promoters pTef1, pTef2 and ADH. On pTUM104 the galactose inducible promoter pGAL1 is located. ]]<br />
<br />
<center>'''Experimental results:'''</center><br><br />
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector backbone via side directed mutagenesis. Furthermore, the original multiple cloning site was replaced by a multiple cloning site compatible with the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for ''in vitro'' applications the new multiple cloning site allows to express proteins with a ''Strep''-tag II. <br />
Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in ''Saccharomyces cerevisiae''.<br />
Moreover, we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities.<br />
<br />
<center>'''Outlook and conclusion:'''</center><br><br />
<br />
The galactose inducible expression system was a great aid for the majority of all subprojects. Especially the opportunity to purify and detect (via Western blot) proteins using the ''Strep''-tag II did facilitate our laboratory practice and accelerated our work progress.<br />
To cover even more demands we are planning to design a second vector template containing a His-tag.<br />
<br />
All BioBricks were sequenced. Sequences can be found in the registry of standard biological parts under the following entries:<br />
<br />
pTUM100 (<partinfo>BBa_K801000</partinfo>), pTUM101 (<partinfo>BBa_K801001</partinfo>), pTUM102 (<partinfo>BBa_K801002</partinfo>), pTUM103 (<partinfo>BBa_K801003</partinfo>) and pTUM104 (<partinfo>BBa_K801004</partinfo>). <br />
<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Vector_Design"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Regulation of Genexpression==<br />
----<br />
By developing inducible promoters and placing them upstream of our biosynthetic pathways we create the possibility to make ''S. cerevisiae'' dynamically respond to concentration changes in its medium as well as to external stimuli. <br />
<br />
An optimal inducing substance needs to be inexpensive, nontoxic and fully controllable in its application. Only substances with these characteristics allow to precisely regulate a system temporally, spatially and quantitatively. <br />
<br />
<br />
<br />
<div class="bezel mfull"><br />
===Ethanol-inducible promoter===<br />
The KlADH4-promoter from the yeast ''Kluyveromyces lactis'' regulates the expression of a mitochondrial alcohol dehydrogenase in an ethanol-dependent way. An alcohol-inducible promoter would be incredibly useful for anyone planning to brew a beer with a transgenic yeast - it would allow for the induction of the target genes after the main fermentation has finished and this way, the metabolic burden for the yeast cells could be lowered. All the transcription factors known to be involved in the regulation of the KlADH4-promoter in ''K. lactis'' also occur in ''S. cerevisiae'' [[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]]. This is why we are confident that this promoter maintains its unique characteristics when transformed into ''S. cerevisiae''.<hr><br />
[[file:TUM12_experiment_overwiew_alcohol1.png|400px|thumb|right| '''A''': The KlADH4-promoter was cloned from genomic DNA of ''Kluyveromyces lactis''. The new BioBrick BBa_K801020 was inserted into our pTUM100 vector. eGFP served as a reporter gene for characterization in ''S. cerevisiae'' (plasmid name: pTUM100_KLADH4_eGFP). '''B''' Emission spectra of eGFP obtained during cultivation of ''S. cerevisiae'' transformed with pTUM100_KLADH4_eGFP using different carbon sources. Blue: Galactose was used as carbon source. The measured ethanol concentration was 1.7 % (v/v). The peak at 509 nm indicates that eGFP is expressed. Red: Glycerol was used as carbon source. The measured ethanol concentration was 0.2 % (v/v). No eGFP fluorescence could be detected.]]<br />
<center>'''Experimental results:'''</center><br><br />
At this time our results concerning the KlADH4-promoter (originally from the yeast ''Kluyveromyces lactis'') suggest that this promoter is ethanol inducible in ''S. cerevisiae''. Further experiments are still being done to abolish residual ambiguities. The lowest ethanol concentration at which eGFP-expression was detected is 0.9 Vol.-%.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
Because ''S. cerevisiae'' is such a good brewer, it was difficult to produce a stringent negative control in our characterization experiments. However, we finally figured out some experiments that allowed us to keep the ethanol concentration below 0.5 % v/v, which is a concentration at which induction is observed in ''K. lactis''. We are working hard on providing additional data, but we are confident that we will be able to provide clear evidence that this promoter is ethanol-inducible not only in ''K. lactis'', but also in ''S. cerevisiae''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Ethanol_Inducible_Promoter"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
<div class="bezel mfull"><br />
<br />
===Light-switchable promoter===<br />
The idea behind a light-switchable system is to create a gene expression system which can be induced and deactivated by light of a certain wavelength.<br />
<br />
This system is extremely attractive, as induction does not require the addition of a specific substance. This makes induction '''cheap, fast, precise''' and also compatible with the Bavarian purity law.<br />
<hr><br />
[[Image:TUM12_lightnew.png|thumb|right|450px|Principle of light-dependent switching of gene-expression.]]<br />
<center>'''Experimental results:'''</center><br><br />
All fusion proteins for the two types of a light-switchable promoter system has been finished ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]), also gene expression batteries coding for all components of each type of our light-switchable promoter system has been done ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]). Since lacking of a second functional yeast vector carrying another auxotrophy marker than URA3 of the pTUM plasmids, which is already reserved for the biosynthesis enzymes, proteins and also reporters, we were not able to clone the whole gene expression battery, into a yeast vector, in order to co-transfect the yeast with one plasmid with the reporter construct and the second plasmid coding for all the devices needed in a light-switchable promoter system.<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
<br />
To get gene expression casette for both of the light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]) into a yeast plasmid, we want to use pSB6A0 ([http://partsregistry.org/Part:BBa_K268000 BBa_K268000]) carrying a TRP1<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Genome Integration==<br />
----<br />
<div class="bezel mfull"><br />
Working with food, it is unacceptable to use antibiotics to keep up the selective pressure during the brewing process. Since we cannot work with auxotrophies in beer either, we have to make sure the yeast cells do not lose the plasmids harboring our BioBricks. The most promising way to accomplish a long lasting presence of our constructs is to achieve <b>genome integration</b>.<br />
<hr><br />
[[file:TUM12_experiment_overwiew_genome.png|500px|thumb|right| Plasmid backbone used for integration of our expression cassettes]]<br />
<center>'''Experimental results:'''</center><br><br />
First experiments to characterize the function of the yeast integration system were performed and the used selection marker was maintained in the yeast culture, although the selection pressure was switched off. This indicates that first integrations were achieved. <br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
Maintaining the plasmids harboring our expression cassettes in the yeast cells during the brewing process is best possible using genome integration. This becomes increasingly interesting, when a yeast strain with different expression cassettes is to be created. Because this is intended for the next step of our project the integration of our expression cassettes becomes increasingly important.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Genome_Integration"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
==Brewing our SynBio Beer==<br />
----<br />
<div class="bezel mfull"><br />
Contrary to popular opinion the chief ingredient of beer is not '''YPD''' but '''gyle''', a carefully prepared mixture of malt, hop and water. Although the name of the yeast strain commonly used in the lab, '''S. cerevisiae''', suggests that it is used in the beer brewing process. The yeast strains generally employed in brewing have '''strongly adapted to gyle''', as they are reutilized in every succeeding brewing cycle.<br />
Hence some investigation on how our yeast '''performs''' in gyle and experiments on the toxicity of the substances produced by our biosynthetic pathways were necessary.<br />
<br><hr><br />
[[file:TUM12_experiment_overwiew_Brewing.png|200px|thumb|right| Picture of the first SynBio Beer brewed during the iGEM competition in 2012]]<br />
<center>'''Experimental results:'''</center><br><br />
Our experiments on the different yeast strains show that the growth of several different yeast strains is '''not impaired in gyle'''! <br><br />
The toxicity test with the substances caffeine and limonene showed a toxicity for yeast cells at higher concentrations in the cultur media. <br><br />
<br />
Expression assays proved the necessity of [[Team:TU_Munich/Project/Genome_Integration|'''genome integration''']] for a proper '''SynBio Beer'''.<br />
<br />
<br />
<center>'''Conclusion and outlook:'''</center><br><br />
At the day of the final Wiki-Freeze we could finish the brewing with three different ingredients (limonene, thaumatin and caffeine) whose biosynthesis we have engineered during this summer. This was done using yeast culture expressing BioBricks from a plasmid as well as using cultures in which the expression cassette was integrated into the genome.<br><br />
This gives us the great honor to present '''iGEM's first and finest SynBio Beer: TUM-Brew'''.<br />
<br />
<div class="noborder" style="float:left"><br />
<html><a href="https://2012.igem.org/Team:TU_Munich/Project/Brewing"><img width="200px" src="https://static.igem.org/mediawiki/2012/3/33/TUM12_readmore.png"/></a></html><br />
</div><br />
</div><br />
<br />
== References ==<br />
----<br />
* [[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10995285 Miranda et al., 2000]] Miranda, C. L., Stevens, J. F., Ivanov, V., McCall, M., Frei, B., Deinzer, M. L., and Buhler, D. R. (2000). Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. ''J Agric Food Chem'', 48(9):3876–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:20:14Z<p>Fabian Froehlich: /* LexA Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
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An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the promoter itself contains a TATA box which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]] Jayaram, M., Li, Y. Y., and Broach, J. R. (1983). The yeast plasmid 2mu circle encodes components required for its high copy propagation. ''Cell'', 34(1):95–104.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:20:04Z<p>Fabian Froehlich: /* GAL4 Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector [[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the promoter itself contains a TATA box which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]] Jayaram, M., Li, Y. Y., and Broach, J. R. (1983). The yeast plasmid 2mu circle encodes components required for its high copy propagation. ''Cell'', 34(1):95–104.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:19:52Z<p>Fabian Froehlich: /* Characterisation via Luciferase Assay */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]], which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the promoter itself contains a TATA box which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]] Jayaram, M., Li, Y. Y., and Broach, J. R. (1983). The yeast plasmid 2mu circle encodes components required for its high copy propagation. ''Cell'', 34(1):95–104.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:19:19Z<p>Fabian Froehlich: /* Reference */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
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</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector, which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the promoter itself contains a TATA box which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6883512 Jayaram et al., 1983]] Jayaram, M., Li, Y. Y., and Broach, J. R. (1983). The yeast plasmid 2mu circle encodes components required for its high copy propagation. ''Cell'', 34(1):95–104.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:12:21Z<p>Fabian Froehlich: /* LexA Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
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An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong pTEF1 promoter and was as well transfected on a high copy vector, which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples. Furthermore investigation of the LexA promoter sequence showed that the promoter itself contains a TATA box which explains the quite high basal expression rate.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter, a low copy plasmid and a different LexA recognition motif, this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:08:49Z<p>Fabian Froehlich: /* GAL4 Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is close to zero.<br />
<br />
As expression the promoter system was driven by the quite strong pTEF1 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong TEF2 promoter and was as well transfected on a high copy vector, which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter and a low copy plasmid this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T01:02:42Z<p>Fabian Froehlich: /* LexA Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
Here the expression the promoter system was as well driven by the quite strong TEF2 promoter and was as well transfected on a high copy vector, which again led to a high number of unspecific bindings of the fusion proteins. Overall the LexA repoter promoter seems to be more sensitive to the concentration of active transcription activating domains, which leads to virtually no difference between the dark, low intensity and high intensity samples.<br />
<br />
Still the normalised RFU is about 10 fold higher than for the GAL4 based system so with a weaker promoter and a low copy plasmid this systems should be a better candidate for a light-switchable system.<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_GAL4_LSPS.pngFile:TUM12 GAL4 LSPS.png2012-10-27T00:55:05Z<p>Fabian Froehlich: uploaded a new version of &quot;File:TUM12 GAL4 LSPS.png&quot;</p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:53:47Z<p>Fabian Froehlich: /* Characterisation via Luciferase Assay */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
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</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
Again PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:52:50Z<p>Fabian Froehlich: /* GAL4 Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 3 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_GAL4_LSPS.pngFile:TUM12 GAL4 LSPS.png2012-10-27T00:52:22Z<p>Fabian Froehlich: uploaded a new version of &quot;File:TUM12 GAL4 LSPS.png&quot;</p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:50:19Z<p>Fabian Froehlich: /* GAL4 Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
<br />
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
<br />
The high intensity sample still shows a 2 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:50:04Z<p>Fabian Froehlich: /* GAL4 Based System */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
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</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
PCB is necessary for correct folding of the PCB-PhyB-DNA-binding-site fusion protein, hence without PCB the output of our reporter system is quite low.<br />
As expression the promoter system was driven by the quite strong TEF2 promoter and was transfected on a high copy vector, there probably was a quite high level of both fusion proteins which led to unspecific binding and a quite high leaky transcription rate for the dark and low intensity samples. This problem could be dealt with by using a weaker promoter or a low copy vector.<br />
The high intensity sample still shows a 2 fold increased induction for a 10 fold increased light intensity compared to the low intensity.<br />
<br />
<br />
<div style="clear:both"><br />
<br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:41:11Z<p>Fabian Froehlich: /* Characterisation via Luciferase Assay */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 7:''' Evaluation of Luciferase Assay for the GAL4 based system.]]<br />
<br />
<div style="clear:both"><br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 8:''' Evaluation of Luciferase Assay for the LexA based system.]]<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
<div style="clear:both"><br />
<br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:39:55Z<p>Fabian Froehlich: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 6:''']]<br />
<br />
<div style="clear:both"><br />
==== LexA Based System ====<br />
</div><br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 7:''']]<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
<div style="clear:both"><br />
=== Outlook for Further Reporter Systems ===<br />
</div><br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:39:11Z<p>Fabian Froehlich: /* Characterisation via Luciferase Assay */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
[[File:TUM12_GAL4_LSPS.png|thumb|right|300px|'''Fig. 6:''']]<br />
<br />
<br />
==== LexA Based System ====<br />
<br />
[[File:TUM12_LexA_LSPS.png|thumb|right|300px|'''Fig. 7:''']]<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Outlook for Further Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_LexA_LSPS.pngFile:TUM12 LexA LSPS.png2012-10-27T00:38:00Z<p>Fabian Froehlich: </p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_GAL4_LSPS.pngFile:TUM12 GAL4 LSPS.png2012-10-27T00:37:48Z<p>Fabian Froehlich: </p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:36:38Z<p>Fabian Froehlich: /* Characterisation via Luciferase Assay */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
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<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
<div style="clear:both"><br />
=== Characterisation via Luciferase Assay ===<br />
</div><br />
<br />
==== GAL4 Based System ====<br />
<br />
==== LexA Based System ====<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Outlook for Further Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:36:03Z<p>Fabian Froehlich: /* Results */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
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An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Extraction of PCB ===<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
=== Characterisation via Luciferase Assay ===<br />
<br />
==== GAL4 Based System ====<br />
<br />
==== LexA Based System ====<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Outlook for Further Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:26:41Z<p>Fabian Froehlich: /* Biosynthesis of Phycocyanobilin */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. As phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
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<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
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An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:24:44Z<p>Fabian Froehlich: /* Light-Switchable Promoter */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be up-regulated as well as down-regulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photo-convertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Systems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-27T00:23:06Z<p>Fabian Froehlich: /* Extraction of PCB */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-27T00:21:03Z<p>Fabian Froehlich: </p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<html><br />
<center><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/51804324?color=d92540" width="800" height="450" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></center></html><br />
<br />
<br />
== Vision ==<br />
----<br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
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<ul id="ticker01" ><br />
<li><span><b>939</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>939</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>939</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
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{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
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<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>Fabian Froehlichhttp://2012.igem.org/Template:Team:TU_Munich/LabHeaderTemplate:Team:TU Munich/LabHeader2012-10-27T00:18:24Z<p>Fabian Froehlich: </p>
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</html></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T20:51:17Z<p>Fabian Froehlich: </p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<html><br />
<center><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/51804324?color=d92540" width="800" height="450" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></center></html><br />
<br />
<br />
== Vision ==<br />
----<br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
</html><br />
<center><br />
<div class="ui-corner-all" id="ticker"><br />
<ul id="ticker01" ><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
</div><br />
</center><br />
<html><br />
<script><br />
$(document).ready(function(){<br />
$('ul#ticker01').liScroll({travelvelocity: 0.03});<br />
});<br />
</script><br />
</html><br />
<br />
{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
<a href="http://www.qiagen.com"><img src="https://static.igem.org/mediawiki/2012/3/39/TUM_Qiuagen.jpg" width="100px"></a><br />
<a href="http://www.carlroth.com"><img src="https://static.igem.org/mediawiki/2012/6/63/TUM_Roth.gif" width="100px"></a><br />
<a href="http://www.bund-der-freunde.tum.de/"><img src="https://static.igem.org/mediawiki/2012/1/1d/TUM_Bund.jpg" width="200px"></a><br />
</center><br />
</html><br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://biologische-chemie.userweb.mwn.de/"><img src="https://static.igem.org/mediawiki/2012/1/1f/TUM_TUM.png" width="400px"></a><br />
</center><br />
</html><br />
<br />
<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T20:50:54Z<p>Fabian Froehlich: </p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<center><br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/51804324?color=d92540" width="800" height="450" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
</center><br />
<br />
== Vision ==<br />
----<br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
</html><br />
<center><br />
<div class="ui-corner-all" id="ticker"><br />
<ul id="ticker01" ><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
</div><br />
</center><br />
<html><br />
<script><br />
$(document).ready(function(){<br />
$('ul#ticker01').liScroll({travelvelocity: 0.03});<br />
});<br />
</script><br />
</html><br />
<br />
{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
<a href="http://www.qiagen.com"><img src="https://static.igem.org/mediawiki/2012/3/39/TUM_Qiuagen.jpg" width="100px"></a><br />
<a href="http://www.carlroth.com"><img src="https://static.igem.org/mediawiki/2012/6/63/TUM_Roth.gif" width="100px"></a><br />
<a href="http://www.bund-der-freunde.tum.de/"><img src="https://static.igem.org/mediawiki/2012/1/1d/TUM_Bund.jpg" width="200px"></a><br />
</center><br />
</html><br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://biologische-chemie.userweb.mwn.de/"><img src="https://static.igem.org/mediawiki/2012/1/1f/TUM_TUM.png" width="400px"></a><br />
</center><br />
</html><br />
<br />
<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_MunichTeam:TU Munich2012-10-26T20:50:27Z<p>Fabian Froehlich: </p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
__NOTOC__<br />
= TUM-Brew: iGEM's first and finest SynBio Beer =<br />
<hr/><br />
<br />
[[File:TUM12_AE120612001.jpg|300px||right||]]<br />
<br />
The TU Munich iGEM Team engineers ''Saccharomyces cerevisiae'', also known as baker's yeast, in order to lay the foundations for a new generation of functional foods with nutritionally valuable ingredients.<br />
<br />
As an example, for iGEM’s first “SynBio Beer” the compounds [[Team:TU_Munich/Project/Xanthohumol|'''xanthohumol''']] (anticancerogenic), [[Team:TU_Munich/Project/Limonene|'''limonene''']] (limeflavor), [[Team:TU_Munich/Project/Caffeine|'''caffeine''']] (CNS-stimulant) as well as the [[Team:TU_Munich/Project/Thaumatin|'''thaumatin''']] (protein sweetener) were chosen to demonstrate the spectrum of possibilities to complement traditional foods or beverages.<br />
<br />
The metabolic pathways for these substances were converted to genetic BioBricks. Using the shuttle vector [[Team:TU_Munich/Project/Vector_Design|'''pTUM100''']], which was adapted to the iGEM standard, transient transfection and expression in yeast were achieved. The gene products were subsequently characterized and their biosynthetic activities investigated.<br />
<br />
[[Team:TU_Munich/Project/Constitutive_Promoter|'''Constitutive''']], [[Team:TU_Munich/Project/Ethanol_Inducible_Promoter|'''ethanol-inducible''']] and [[Team:TU_Munich/Project/Light_Switchable_Promoter|'''light-switchable''']] promoter systems were developed to individually regulate the expression of these gene cassettes. By combining these BioBricks our team has been able to brew iGEM’s first and finest [[Team:TU_Munich/Project/Brewing|'''SynBio Beer''']].<br />
<br />
<html><iframe style="box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);padding: 5px;margin: 5px;background-color: white;" src="http://player.vimeo.com/video/51804324?color=d92540" width="800" height="450" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe></html><br />
<br />
== Vision ==<br />
----<br />
<br />
We, TU Munich’s 2012 iGEM team, strive to catalyze the diffusion process of knowledge about genetic engineering and synthetic biology among the general public. Using the example of iGEM’s first and finest SynBio Beer we involve, interest and inspire people to reconsider preconceived ideas and encourage them to openly engage in a broad discussion weighing pros and cons of genetic engineering in foodstuff. We sketch a future where new technology can be applied in a meaningful way to complement traditional foods or beverages.<br />
<br />
== Overview ==<br />
----<br />
<html><br />
<script src="https://2012.igem.org/Team:TU_Munich/jquery.li-scroller.1.0.js?action=raw&ctype=text/js"></script><br />
</html><br />
<center><br />
<div class="ui-corner-all" id="ticker"><br />
<ul id="ticker01" ><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
<li><span><b>878</b> Documented experiments in our labjournal</span></li><br />
<li><span><b>923</b> Plasmids</span></li><br />
<li><span><b>49</b> Annotated BioBricks</span></li><br />
<li><span><b>107</b> Days in the lab</span></li><br />
<li><span><b>283</b> Sequencing orders</span></li><br />
<li><span><b>13</b> Subprojects</span></li><br />
<li><span><b>19</b> Students</span></li><br />
<li><span>Over <b>10.000.000</b> systems of differential equations solved</span></li><br />
</ul><br />
</div><br />
</center><br />
<html><br />
<script><br />
$(document).ready(function(){<br />
$('ul#ticker01').liScroll({travelvelocity: 0.03});<br />
});<br />
</script><br />
</html><br />
<br />
{{Team:TU_Munich/Overview}}<br />
<br />
== Reference ==<br />
----<br />
<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984]] Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. ''Cell'', 37(2):629–33.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10724480 Mazzoni et al., 2000]] Mazzoni, C., Santori, F., Saliola, M., and Falcone, C. (2000). Molecular analysis of uas(e), a cis element containing stress response elements responsible for ethanol induction of the kladh4 gene of ''Kluyveromyces lactis''. ''Res Microbiol'', 151(1):19–28.<br />
<br />
== Sponsors ==<br />
----<br />
<br />
<html><br />
<center class="noborder"><br />
<a href="http://www.applichem.com"><img src="https://static.igem.org/mediawiki/2012/f/f5/TUM_Applichem.jpg" width="200px"></a><br />
<a href="http://www.biomers.net"><img src="https://static.igem.org/mediawiki/2012/3/37/TUM_Biomers.png" width="200px"></a><br />
<a href="http://www.biozym.com"><img src="https://static.igem.org/mediawiki/2012/d/d8/TUM_Biozym.jpg" width="200px"></a><br />
<a href="http://www.cipsm.de"><img src="https://static.igem.org/mediawiki/2012/8/88/TUM_Cipsm.jpg" width="200px"></a><br />
<a href="http://www.eurofins.de"><img src="https://static.igem.org/mediawiki/2012/b/bc/TUM_Eurofins.png" width="200px"></a><br />
<a href="http://www.idt.com"><img src="https://static.igem.org/mediawiki/2012/0/0b/TUM_IDT.jpg" width="200px"></a><br />
<a href="http://www.metabion.com"><img src="https://static.igem.org/mediawiki/2012/9/93/TUM_Metabion.png" width="200px"></a><br />
<a href="http://www.geneious.com/"><img src="https://static.igem.org/mediawiki/2012/8/82/TUM_Geneious.png" width="200px"></a><br />
<a href="http://www.roche.de/"><img src="https://static.igem.org/mediawiki/2012/a/a4/TUM_Roche.jpg" width="200px"></a><br />
<a href="http://www.daad.de/promos/"><img src="https://static.igem.org/mediawiki/2012/7/77/TUM_DAAD.gif" width="200px"></a><br />
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<center>http://www4.clustrmaps.com/stats/maps-no_clusters/2012.igem.org-Team-TU_Munich-thumb.jpg</center></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox4.JPGFile:TUM12 Lightbox4.JPG2012-10-26T20:43:31Z<p>Fabian Froehlich: uploaded a new version of &quot;File:TUM12 Lightbox4.JPG&quot;</p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox3.JPGFile:TUM12 Lightbox3.JPG2012-10-26T20:43:05Z<p>Fabian Froehlich: uploaded a new version of &quot;File:TUM12 Lightbox3.JPG&quot;</p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox1.JPGFile:TUM12 Lightbox1.JPG2012-10-26T20:39:40Z<p>Fabian Froehlich: uploaded a new version of &quot;File:TUM12 Lightbox1.JPG&quot;</p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:33:04Z<p>Fabian Froehlich: /* Extraction of PCB */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
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An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|400px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|400px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:32:45Z<p>Fabian Froehlich: /* Extraction of PCB */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|420px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|420px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:32:32Z<p>Fabian Froehlich: /* Extraction of PCB */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|450px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|right|450px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:32:06Z<p>Fabian Froehlich: /* Extraction of PCB */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|thumb|right|250px]]<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|'''Fig. 5:''' Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|'''Fig. 6:''' Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:30:52Z<p>Fabian Froehlich: /* Components of the Light-Switchable Promoter Systems */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb|'''Fig. 4:''' Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox1.JPGFile:TUM12 Lightbox1.JPG2012-10-26T20:29:53Z<p>Fabian Froehlich: uploaded a new version of &quot;File:TUM12 Lightbox1.JPG&quot;</p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox1.JPGFile:TUM12 Lightbox1.JPG2012-10-26T20:28:17Z<p>Fabian Froehlich: </p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:26:25Z<p>Fabian Froehlich: /* Induction Setup */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|200px]]<br />
[[File:TUM12_Lightbox2.JPG|200px]]<br />
[[File:TUM12_Lightbox3.JPG|200px]]<br />
[[File:TUM12_Lightbox4.JPG|200px]]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb| Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:25:14Z<p>Fabian Froehlich: /* Induction Setup */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|thumb|200px|'''Fig. 4:''']]<br />
[[File:TUM12_Lightbox2.JPG|thumb|200px|'''Fig. 5:''']]<br />
[[File:TUM12_Lightbox3.JPG|thumb|200px|'''Fig. 6:''']]<br />
[[File:TUM12_Lightbox4.JPG|thumb|200px|'''Fig. 7:''']]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb| Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:24:48Z<p>Fabian Froehlich: /* Induction Setup */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox1.JPG|thumb|200px|center|'''Fig. 4:''']]<br />
[[File:TUM12_Lightbox2.JPG|thumb|200px|center|'''Fig. 5:''']]<br />
[[File:TUM12_Lightbox3.JPG|thumb|200px|center|'''Fig. 6:''']]<br />
[[File:TUM12_Lightbox4.JPG|thumb|200px|center|'''Fig. 7:''']]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb| Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:21:41Z<p>Fabian Froehlich: /* Induction Setup */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO micro-controller that puts the suggested protocol.<br />
The use of a micro-controller will allow us to easily test different pulse lengths and frequencies.<br />
<br />
<center><br />
[[File:TUM12_Lightbox2.JPG|thumb|200px||'''Fig. 4:''']]<br />
[[File:TUM12_Lightbox2.JPG|thumb|200px||'''Fig. 5:''']]<br />
[[File:TUM12_Lightbox2.JPG|thumb|200px||'''Fig. 6:''']]<br />
[[File:TUM12_Lightbox2.JPG|thumb|200px||'''Fig. 7:''']]<br />
</center><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb| Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox4.JPGFile:TUM12 Lightbox4.JPG2012-10-26T20:20:32Z<p>Fabian Froehlich: </p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox3.JPGFile:TUM12 Lightbox3.JPG2012-10-26T20:20:25Z<p>Fabian Froehlich: </p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/File:TUM12_Lightbox2.JPGFile:TUM12 Lightbox2.JPG2012-10-26T20:20:05Z<p>Fabian Froehlich: </p>
<hr />
<div></div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:18:34Z<p>Fabian Froehlich: /* Induction Setup */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<!--<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
--><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO microcontroller that realizes the suggested protocol.<br />
The use of a microcontroller will allow us to easily test differrent pulse lengths and frequencys.<br />
<br />
<br><br />
<br><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb| Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
<br />
[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlichhttp://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_PromoterTeam:TU Munich/Project/Light Switchable Promoter2012-10-26T20:01:07Z<p>Fabian Froehlich: /* Biosynthesis of Phycocyanobilin */</p>
<hr />
<div>{{Team:TU_Munich/Header}}<br />
= Light-Switchable Promoter =<br />
----<br />
<br />
[[File:Jeff_einzel_TUM12.jpg|200px|thumb||Responsible: Jeffery Truong]]<br />
<br />
<div style="text-align:justify;"><br />
The so-called "Reinheitsgebot" or "Bavarian Beer Purity Law" forbids the use of any ingredients other than water, barley and hops.<br />
Hence, to be able to control the expression of our pathways in yeast, a promoter which does not rely on any chemical additive.<br />
<br />
The light switchable promoter, does not only comply with these needs, it is also easy, cheap and very precisely applicable.<br />
Furthermore, as the expression of the downstream gene can be upregulated as well as downregulated by variation of red light and far red light ratio respectively.<br />
<br />
Therefore it allows high spatio-temporal control over the genes downstream of the promoter.<br />
<br><br />
<br><br />
<br><br />
<br />
==Background and Principles==<br />
----<br />
This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e.&nbsp;g. LacZ or an auxotrophic marker.<br />
<br />
=== Reverse Yeast-Two Hybrid Based Light-Switchable Promoter System ===<br />
<br />
This basic principle is utilized in the yeast light-switchable promoter system. But in contrast to yeast-two hybrid, we already know the interaction partners (PhyB and PIF3). The photoconvertible binding of PhyB to PIF3 is used, to recover the physical contiguity of the DNA binding domain and the transcriptional activation domain under defined conditions (red light).<br />
<br />
<br />
[[Image:TUM12_light.jpg|thumb|right|300px|'''Fig. 1''' Principle of light-dependent switching of gene-expression.]]<br />
This light-inducible system contains two proteins, phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3). PhyB and PIF3 will just form a heterodimer, if PhyB is exposed to red light. Exposition under red light leads to a conformation change of PhyB to its active form (P<sub>fr</sub>-form); the P<sub>fr</sub> form of PhyB now can bind PIF3. PhyB comprises a light-absorbing chromophore phycocyanobilin, which gives PhyB the ability to undergo a photoconversion to the active P<sub>fr</sub> form (red light exposition) or back to its ground-state P<sub>r</sub> (far-red light exposition or darkness).<br />
<br />
==== GAL4 Based Light-Switchable Promoter System ====<br />
<br />
In our first case we create two constitutively expressed fusion proteins, the first one is PhyB fused to GAL4DBD for the DNA binding part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] and the second one is PIF3 fused to GAL4AD for the transcriptional activating part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]). This system allows us to control spatio-temporally the expression of our genes coded on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801004 pTUM104] and driven by the GAL1 promoter (The TATA-box of pGAL1 is preceded by binding elements for GAL4). To prevent interference with the endogenous GAL4 system of yeast, we are using the Y190 ''S.&nbsp;cerevisiae'' strain, which has an GAL4/GAL80 deletion.<br />
<br />
One great advantage of the GAL4 based system is that we can use all our constructs which we have first cloned downstream of a GAL1 promoter without further cloning steps! But the disadvantage is that we have to use a yeast strain carrying a GAL4/GAL80 deletion.<br />
<br />
If you want to use a supermarket yeast or a brewing strain you have to use the LexA based light-switchable promoter system, described in the next section.<br />
<br />
==== LexA Based Light-Switchable-Promoter System ====<br />
<br />
In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.<br />
<br />
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites, e.&nbsp;g. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K165031 BBa_K165031].<br />
<br />
In distinction from the GAL4 based system there is no necessity for a special strain carrying an GAL4/80 deletion, so theoretically every yeast strain can be used for this system.<br />
<br />
=== Biosynthesis of Phycocyanobilin ===<br />
Phycocyanobilin undergoes a Z-E isomerization to its active form in case of red light and an E-Z isomerization to its inactive form in case of far-red light. The half-life of its active form P<sub>fr</sub> is ~30 min, so continuous red light exposition is not necessary. A great advantage is that light-sensitive odorant and flavorings will not be destroyed. Once phycocyanobilin is not naturally available in yeast one have to add the tetrapyrrole light-absorbing chromophore phycocyanobilin to the medium to get a functional light-switchable promoter system. But it also possible to bring the capability of phycocyanobilin synthesis in yeast by metabolic engineering. From heme, which is endogenous in yeast, there are only two steps of biosynthesis away from phycocyanobilin. The first step of phycocyanoblin is catalyzed by a heme oxygenase, the second step by a phycocyanobilin:ferredoxin oxidoreductase.<br />
<br />
[[Image:TUM12 PCB synthesis.jpg|thumb|left|400px|'''Fig. 2:''' Biosynthesis pathway of phycocyanobilin from heme to phycocyanobilin (PCB).]]<br />
<br />
[[Image:TUM12 modelling PCB binding cavity PhyB.jpg|thumb|left|400px|'''Fig. 3:''' Cavity of PCB binding pocket of PhyB, predicted by I-TASSER. The next most homologue protein is illustrated in cyan, the cyanobacterial phytochrome CPH1 [http://www.rcsb.org/pdb/explore.do?structureId=2VEA 2VEA]. The golden ribbon indicates the predicted structure of PhyB. The sulfhydryl group of the ''Arabidopsis'' chromophore-binding cysteine residue is co-ordinated with the position of the ethylidene moiety on the chromophore sufficiently closely and in the correct conformation to form the thioether bond by which the chromophore is known to be covalently attached.]]<br />
<br />
=== Induction Setup ===<br />
<html><br />
<iframe style="float:right;padding:5px;margin:10px;box-shadow: 1px 1px 2px rgba(0, 0, 0, 0.2);" width="300" height="200" src="http://www.youtube.com/embed/CbN-ObW0K3I" frameborder="0" allowfullscreen></iframe><br />
</html><br />
An array of 10 LEDs with emission peak at 660&nbsp;nm [http://www.alldatasheet.com/datasheet-pdf/pdf/296270/ROITHNER/B5-436-30D.html] were attached into the molds of the packaging of 2&nbsp;ml cuvettes and soldered together on the rear side of the packaging. As the cuvettes are the very ones that will later be used for illumination of the cells, the use of the packaging as LED matrix will allow quick removal during measurements and enhance accuracy of results.<br />
<br />
Literature suggest pulsed illumination of the cells with a pulse duration of 10&nbsp; and a pulse frequency of 1 pulse every 10 minutes. The LEDs are actuated with an Arduino UNO microcontroller that realizes the suggested protocol.<br />
The use of a microcontroller will allow us to easily test differrent pulse lengths and frequencys.<br />
<br />
<br><br />
<br><br />
<br />
== Results ==<br />
----<br />
=== Components of the Light-Switchable Promoter Systems ===<br />
<br />
Two fusion proteins will be needed for a light-switchable promoter system. The first one is PIF3 fused to GAL4AD ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039]), the second one is GALDBD (GAL4 based) or LexA (LexA based) fused to PhyB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041]).<br />
<br />
For PhyB and PIF3 we didn't used the whole protein coding sequence for our fusions. For PhyB we used the first 908 N-terminal amino acids which has been mapped to be sufficient for reversible photoconversion. Also for PIF3 only the first 100 N-terminal amino acids has been taken for our fusions due to the fact that they has been mapped to be only necessary for light-switchable binding to PhyB.<br />
<br />
We successfully created all fusion proteins for a light-switchable promoter system based on GAL4 and LexA and even created a TEF1 promoter driven expression battery for all our components, for each type of the system (GAL4 and LexA based).<br />
<br />
[[file:TUM12_JeffscloningIII.png|900px|right|thumb| Simplified cloning scheme for the GAL4 ('''A''') and the LexA ('''B''') based gene expression battery.]]<br />
<br />
* Fusion protein for the first component (GAL4/LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801039 BBa_K801039: SV40NLS-GAL4AD-Linker-PIF3]<br />
<br />
* Fusion protein for the second component (GAL4 based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801040 BBa_K801040: SV40NLS-PhyB-Linker-GAL4DBD]<br />
<br />
* Fusion protein for the second component (LexA based):<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801041 BBa_K801041: SV40NLS-PhyB-Linker-LexA]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the GAL4 based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4DBD_tTEF1]<br />
<br />
* TEF1 promoter driven gene expression battery for all parts of the LexA based light-switchable-promoter system:<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043: pTEF1_SV40NLS-GAL4AD-Linker-PIF3_tTEF1_pTEF1_SV40NLS-PhyB-Linker-GAL4LexA_tTEF1]<br />
<br />
=== Components for Reporter Systems ===<br />
<br />
==== GAL4 Based Reporter Rystems ====<br />
For the GAL4 based light-switchable promoter system we have endogenous reporters in the Y190 ''S.&nbsp;cerevisiae'' strain.<br />
<br />
The first one is an auxotrophic reporter for HIS3, an imidazoleglycerol-phosphate dehydratase, which catalyzes the sixth step in histidine biosynthesis. HIS3 is driven by a synthetic promoter with upstream GAL4 responsive elements. If plated on or inoculated in histidine deficient medium, there should be no growth of yeast, if they will be incubated in darkness or far-red light conditions. But under red light conditions the auxotrophy is reverted by expression of HIS3 due to the recruitment of GAL4AD through PhyB-PIF3 interaction.<br />
<br />
The second reporter is LacZ, a beta-galactosidase, which will be controlled by pGAL1. Beta-galactosidase will be only expressed, if the light-switchable promoter system is switched on by red light.<br />
<br />
==== LexA Based Reporter Systems ====<br />
<br />
For the LexA based light-switchable promoter system we have to transfect yeast with a second plasmid coding for the reporter construct because there is no endogenous reporter system like for the GAL4 based system. Furthermore we didn't used the GAL4/GAL80 deletion strain Y190 in contrast to the GAL4 based system, since there is no need for the deletion because there is no interference between the prokaryotic LexA system the endogenous yeast signaling and the metabolism pathways.<br />
<br />
We've successfully cloned a luciferase from ''Renilla&nbsp;reniformis'' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_J52008]) downstream of a minimal CYC1 promoter preceded by LexA binding sites ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J52008 BBa_K165031]).<br />
<br />
=== Extraction of PCB ===<br />
<br />
Since there is no endogenous phycocyanobilin (PCB) in yeast, we have to add it to the medium first for our first proof-of-concept experiments. Later, we can implement the enzymes for the biosynthesis of phycocyanobilin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15008 BBa_I15008] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K181000 BBa_K181000]) also in the finished gene expression batteries for our light-switchable promoter systems([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801042 BBa_K801042] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K801043 BBa_K801043]).<br />
<br />
* Phycocyanobilin is extracted by methanolysis of dried ''Spirulina&nbsp;platensis''. For detailed information please see our [https://2012.igem.org/Team:TU_Munich/Notebook/Protocols methods] section<br />
<br />
* The extracted phycocyanobilin is resuspended in DMSO and is kept at -20&nbsp;°C until use.<br />
<br />
* Absorption Spectrum for concentration determination.<br />
<br />
[[Image:TUM12_20120920_PCB_absorptionspectrum.jpg|thumb|left|500px|Absorption spectrum of the extracted phycocyanobilin]]<br />
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[[File:TUM12 formula PCBconc determination.jpg|250px]]<br />
<br />
[[Image:TUM12_LSPS_WP_000734.jpg|thumb|left|500px|Sample of the phyocyanobilin colloid]]<br />
<br />
== Reference ==<br />
----<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005]] Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. ''Curr Biol'', 15(7):637–42.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009]] Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. ''PLoS Genet'', 5(1):e1000352.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009]] Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. ''Nature'', 461(7266):997–1001.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002]] Mendelsohn, A. R. (2002). An enlightened genetic switch. ''Nat Biotechnol'', 20(10):985–7.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002]] Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. ''Nat Biotechnol'', 20(10):1041–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004]] Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. ''Plant Cell'', 16(11):3033–44.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001]] Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. ''Proc Natl Acad Sci U S A'', 98(19):10566–71.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999]] Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. ''Nature'', 400(6746):781–4.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999]] Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. ''Biol Proced Online'', 2:1–38.<br />
*[[http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985]] Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of ''Escherichia coli'' K-12. ''J Bacteriol'', 163(1):376–84.</div>Fabian Froehlich