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| − | <h2 id="MembraneIntegration">Membrane Integration</h2>
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| − | <p>To optimize the luminal conditions of our compartment we focused on integrating new proteins to its membrane. This way we can alter specific properties or supply reactions inside with necessary co-factors. To test the import and integration mechanism, we fused our designed membrane anchors to fluorescent marker proteins and finally integrated the protein pump bacteriorhodopsin into the membrane to acidify our compartment</p>
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| − | <h3>Introduction</h3>
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| − | < p> Many reactions rely on optimal conditions like pH and co-factors. Thus, this subproject aims at the optimization of those circumstances through the integration of new membrane proteins, which alter specific properties of the peroxisomal lumen. Such an approach promises to be very useful for metabolic engineering projects as it can help to adjust the pH, provide cofactors to enzymes or increase/decrease the concentrations of metabolites inside to peroxisome. In nature two distinct mechanisms exist, which are used for the integration of membrane proteins into the peroxisomal membrane – a Pex19-<a href=" http:// www. uniprot.org/ uniprot/ P28795"> Pex3</ a> dependent and an ER-dependent one < a href="http://www.plantphysiol.org/content/ 139/2/690"> | + | <div class="callout"> |
| − | <abbr title="(Alison Baker et al.)">.</p>
| + | <table> |
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| | + | <th><h3 style="color:rgb(0, 0, 0)"><a style="color:rgb(0,0,0)" href=" https:// 2017. igem.org/ Team:Cologne-Duesseldorf/ Results"> Results</ h3></ th> |
| | + | <th><h3 style="color:rgb(0,0,0)">Achieved</h3></th> |
| | + | </tr> |
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| − | <img src="https://static.igem.org/mediawiki/2017/8/8c/PMP_pH_dependent_enzymes.png"> | + | <tr> |
| | + | <td>We were able to design and successfully test an orthogonal peroxisomal protein <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#PEX5Import">import mechanism</a> for peroxisomes in <i>S. cerevisiae<i></td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/ 4/ 46/ T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | < p> They rely on a so called mPTS sequence, that is used to mark the proteins for transport to and integration in the peroxisomal membrane <a href="https:// www. ncbi. nlm.nih.gov/ pubmed/ 12839494"> | + | <tr> |
| − | <abbr title="(2003, H.F. Tabak et al.)">. We will try to utilize the capability of both mechanisms to incorporate new proteins into the peroxisomal membrane.
| + | <td>By decorating the peroxisomes with the v-SNARE Snc1 we successfully <a href="https:// 2017. igem. org/ Team:Cologne-Duesseldorf/ Results#Secretion" >secreted</a> their entire contents</td> |
| − | However, to test whether yeast can integrate and use the foreign proteins in its peroxisomal membrane, we will design three different constructs, which will hopefully give us insights into the mechanisms and its efficiency to incorporate new proteins into the peroxisomal membrane.</p>
| + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| | + | <tr> |
| | + | <td>With two different <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#Sensors">sensors</a> we were able to efficiently measure the pH and the redox potential inside our yeast peroxisomes</td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | <img src="https://static.igem.org/mediawiki/2017/7/7b/PMP_Import_ways.png"> | + | <tr> |
| | + | <td>Via fluorescence microscopy we verified that the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#MembraneIntegration">integration of new membrane proteins</a> into the peroxisomal membrane is possible</td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/ 4/ 46/ T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| | + | <tr> |
| | + | <td>By successfully translocating the required enzymes for the metabolic pathways of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#Nootkatone">Nootkatone</a> and <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#Violacein">Violacein</a> into the peroxisome and actually synthesizing the latter, we developed a proof of concept for our toolbox</td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| | + | <td>We successfully implemented a way of customizing the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results#SizeAndNumber">size and number</a> of the peroxisomes into our toolbox</td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | < p> As a proof of concept, we will incorporate three proteins through three different approaches into the peroxisomal membrane: (i) mRuby2-<a href=" http:// www. uniprot.org/ uniprot/Q7Z412"><a href="http:/ /www.uniprot.org/uniprot/Q7Z412" style="color:# DB8321"> PEX26</a></ a> as a proof for the Pex19-dependent mechanism, (ii) < a href=" http:// www. uniprot.org/ uniprot/ P28795">Pex3</ a>- mRuby2 itself to showcase the ER- dependent mechanism and (iii) <a href=" http://www.uniprot.org/uniprot/P02945" >bacteriorhodopsin</a>, a unidirectional proton pump, fused to the N-terminal anchor of <a href=" http://www.uniprot.org/uniprot/P28795"> Pex3</ a> . </ p> | + | <tr> |
| | + | <td>With a high throughput assay we characterized the import efficiency of <a href=" https:// 2017. igem.org/ Team: Cologne-Duesseldorf/ Results# Pex7Import"> different PTS2 sequences</a></ td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | <h4>Pex19-dependent Mechanism</h4> | + | <tr> |
| | + | <td>To get a better understanding of possible problems and pitfalls of our metabolic engineering concepts we extensively <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Model">modeled</a> the whole nootkatone pathway and the benefits of it being translocated inside our compartment</td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | < p> The exact mechanisms of mPTS binding, < a href="http://www.uniprot.org/uniprot/P28795"> Pex3</a>/Pex19 disassembly, mPTS-PMP binding, and release from the <a href=" http:// www. uniprot.org/ uniprot/ P28795"> Pex3</a> /Pex19 mediated mPTS-PMP docking to the full integration into the membrane are yet unknown < a href="https:/ /www.ncbi.nlm.nih.gov/pubmed/20531392"> | + | <tr> |
| − | <abbr title="(2010,Schueller et al.)">[4]. However, general principles of the integration of a new peroxisomal membrane protein (PMP) through Pex19 and <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> are studied. Most PMPs feature a membrane targeting signal (mPTS), multiple binding sites for Pex19p, and at least one transmembrane domain (TMD). The mPTS can appear in two different ways, either located in the middle of the primary amino acid sequence, which is the rather complex form, or it can be found at the N-terminal part of the PMP as in Pex25.Pex19p is a cytosolic protein, which recognizes the mPTS of the PMP to be incorporated. In the first step Pex19p attaches to the PMP by binding to the mPTS and acts like a chaperone, guiding it to the peroxisome. Next, Pex19p binds N-terminally to the peroxisomal membrane protein <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>p, which is attached to the peroxisomal membrane through an N-terminal membrane anchor. This will bring the PMP in close proximity to the peroxisomal membrane. Last, Pex19p initiates the membrane integration of the PMP. <a href="https://www.ncbi.nlm.nih.gov/pubmed/26777132">
| + | < td> For our planned optogenetic experiments we designed an affordable <a href=" https:// 2017. igem.org/ Team:Cologne-Duesseldorf/ Hardware"> lightbox</a> which can easily be assembled in a short time |
| − | <abbr title="(2016, Liu et al)">.</p>
| + | </td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | <h4>Additional Sources/References</h4> | + | <tr> |
| | + | <td>All our excellent <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Results">results</a> can be combined into a highly variable compartment <strong>toolbox</strong> for designing artificial compartments based on the peroxisomes in <i>S. cerevisiae</i> with an enormous range of applications |
| | + | </td> |
| | + | <td><img src="https://static.igem.org/mediawiki/2017/4/46/T--Cologne-Duesseldorf--check.png" width="100" height="100"></td> |
| | + | </tr> |
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| − | <p>2001, Jones - Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins</p>
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| − | <p>2004, Jones - PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins</p>
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| − | <p>2004, Rottensteiner - Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals</p>
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| − | <p>2016, Mayerhofer - Targeting and insertion of peroxisomal membrane proteins ER trafficking versus direct delivery to peroxisomes</p>
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| − | <p>2016, Hua - Multiple paths to peroxisomes Mechanism of peroxisome maintenance in mammals</p>
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| − | <p>2016, Giannopoulou - Towards the molecular mechanism of the integration of peroxisomal membrane proteins</p>
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| | + | </tbody> |
| | + | </table> |
| | </div> | | </div> |
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