Difference between revisions of "Team:Cologne-Duesseldorf/Banner"

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              <td>OMG</td>
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              <td>WTF</td>
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<td><div><a href="https://2017.igem.org/Team:Aix-Marseille ">MAX MUSTERMANN</a> align="right"</div></td>
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<td>SCHEIßE</td>
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<td>SCHEIßE2</td>
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<td><a href="https://2017.igem.org/Team:Aachen">Aachen</a></td>
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<td><a href="https://2017.igem.org/Team:Aalto-Helsinki/Collaborations">Aalto-Helsinki</a></td>
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<td><a href="https://2017.igem.org/Team:Aix-Marseille ">Aix-Marseille</a></td>
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<td><a href="https://2017.igem.org/Team:Bielefeld-CeBiTec ">Bielefeld-CeBiTec</a></td>
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<td><a href="https://2017.igem.org/Team:UBonn_HBRS ">UBonn_HBRS</a></td>
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<td><a href="https://2017.igem.org/Team:ColumbiaNYC">ColumbiaNYC</a></td>
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        <td>HIER DEN TEXT</td>
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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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255988"> <abbr title="I.A. Sparkes, C. Hawes, A. Baker, AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes, Plant Physiol. 139 (2005) 690–700"> (2005, Sparkes <i>et al.</i>) </abbr> </a>.</p>
 
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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"> <abbr title="H.F. Tabak, J.L. Murk, I. Braakman, H.J. Geuze, Peroxisomes start their life in the endoplasmic reticulum, Traffic 4 (2003) 512–518"> (2003, H.F. Tabak <i>et al.</i>)</abbr> </a>. We will try to utilize the capability of both mechanisms to incorporate new proteins into the peroxisomal membrane. 
 
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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>
 
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<img src="https://static.igem.org/mediawiki/2017/7/7b/PMP_Import_ways.png">
 
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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>
 
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<h4>Pex19-dependent Mechanism</h4>
 
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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"> <abbr title="2010, Schueller - The peroxisomal receptor Pex19p forms a helical mPTS recognition domain"> (2010, Schueller <i>et al.</i>)</abbr> </a>. 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">
 
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  <abbr title="(2016, Liu et al)">.</a></p>
 
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<h4>Additional Sources/References</h4>
 
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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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  <h2 id=”Membrane_Integration”>Membrane integration</h2>
 
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  <p>Abstract</p>
 
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<p>Peroxisomal membrane proteins are synthesized on free polysomes in the cytosol and afterwards integrated into the membrane via two major pathways: one dependent on the endoplasmatic reticulum and one dependent on PEX19 and PEX3.
 
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<h3>Experimental Work/Design</h3>
 
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<p>In order to test our hypothesis we fused the last 59 amino acids of the C-terminus of human <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> (AA 246-305) to a red fluorescent protein, to further elucidate the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>/Pex19-dependent import. mRuby is generally used as a marker in combination with a fluorescent microscope to visualize the localization of the fusion protein. The C-terminus of <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> contains a helical signal-anchor, which serves as both, a mPTS and transmembrane domain. We designed our construct with mRuby2 fused to the N-terminal side of the <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a>-C-terminus, this way the mRuby should face the cytosolic side of the peroxisomal membrane. 
 
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Quite similar to our mRuby-<a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> approach, we designed a construct for the ER-dependent import. Therefore, we fused the mRuby2 fluorescent protein to the N-terminus of  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> (AA 1-39). This construct should be N-terminally anchored in the peroxisomal membrane, with mRuby2 again facing the cellular lumen.</p>
 
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<p>Our main goal is to introduce a rather complex membrane protein to the peroxisome that can alter specific traits. For that we fused the  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> N-terminus (AA 1-39) to a <i>Halobacterium salinarum</i> <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> protein (AA 16-262), replacing the first 16 amino acids (<a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-BacR). The original archaeal <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> acts as a proton pump by capturing light energy to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy.
 
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Our assumption is that the first transmembrane segment determines the orientation of the following protein and that therefore due to the N-terminal anchoring signal the <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> will be inserted in reverse orientation, pumping the protons into the peroxisome. This way the pH of the peroxisomal lumen could actively be controlled and adjusted.</p>
 
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<p>Finally, our project involved combining the work of other subteams to verify the localization of our constructs in the peroxisome and analyze the effects they have on the import. Therefore, we are using the superfolded-GFP protein, another fluorescent marker, which is in our case fused to the peroxisomal import sequence PT1, and a version of Pex11 that is fused to the fluorescent marker Venus. Both markers emit light in the green light spectrum, were as mRuby2 emits light in the red part of the spectrum, giving us a strong contrast and an easy way of differentiating between the two under the fluorescent microscope.  </p>
 
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<p>To physically create our constructs, we researched the DNA sequences of <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>,  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> via UniProt and pre-designed our fusion constructs with the software tool „Geneious“. We ordered the synthesis of three separate parts ( <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>, <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> and  <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>-BacR) from IDT. To ease out the process of assembling our plasmids, we used the „<a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#h2-2">Dueber Toolbox</a>", containing various parts such as promoters and terminators, to tailor the plasmids specific to your needs. Finally, to combine all the selected parts, we used the „Golden Gate” assembly method.</p>
 
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<p>In order to have full control over the amount of expressed protein, we designed our plasmids with the inducible galactose promoter "pGAL1". Not only were we able to see that our fluorescent marked protein anchors from <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a>  and  <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> would localize at specific points inside our cells but also to show that it was in deed the peroxisome they were accumulating at. For that we coexpressed each of our fluorescent membrane anchors together with a GFP protein that was fused to a PTS1 sequence and thus imported into the peroxisome.Fluorescent microscopy was used to colocalize both, the green fluorescing GFP and the red fluorescing mRuby and it is clearly visible, that our anchors integrated into the peroxisomal membrane.</p>
 
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Finally we used the same approach to direct a mRuby-tagged <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> to our compartment. In coexpressing it with the same GFP as in the previous steps, we could show that the <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> as well as <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> were successfully integrated into the membrane of our compartment. Since <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> is a rather complex protein, we're very optimistic about integrating other proteins into the membrane using the same approach.
 
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<h4>Outlook</h4>
 
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<p>The ultimate goal of this subproject is, to have a complete set of ready to transform membrane proteins that could be combined with any promoter to create the optimal conditions for each desired situation. Besides <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>, we also started to work with sugar translocators, since yeast does not posses the ability to import it into or export it from the peroxisome. This would open up a whole new chapter of peroxisomal usage, from example as a temporary storage compartment.
 
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Revision as of 19:45, 1 November 2017

Testheading

OMG WTF
MAX MUSTERMANN align="right"
 SCHEIßE SCHEIßE2
Aachen Aalto-Helsinki Aix-Marseille
Bielefeld-CeBiTec UBonn_HBRS ColumbiaNYC
HIER DEN TEXT

Results and engineered constructs of artico

  • We were able to design and successfully test an orthogonal peroxisomal protein import mechanism for the peroxisome in S. cerevisiae.
  • By decorating the peroxisomes with the v-SNARE Snc1 we successfully secreted their entire contents
  • With two different sensors we were able to efficiently measure the pH and the redox potential inside our yeast peroxisomes.
  • Via fluorescence microscopy we verified that the integration of new membrane proteins into the peroxisomal membrane is possible.
  • By successfully translocating the required enzymes for the metabolic pathways of Nootkatone and Violacein into the peroxisome and actually synthesizing the latter, we developed a proof of concept for our toolbox
  • We successfully implemented a way of customizing the size and number of the peroxisomes into our toolbox.
  • With a high throughput assay we characterized the import efficiency of different PTS2 sequences.
  • To get a better understanding of possible problems and pitfalls of our metabolic engineering concepts we extensively modeled the whole nootkatone pathway and the benefits of it being translocated inside our compartment.
  • For our planned optogenetic experiments we designed an affordable lightbox which can easily be assembled in a short time.
  • All our excellent results can be combined into a highly variable compartment toolbox for designing artificial compartments based on the peroxisomes in S. cerevisiae with an enormous range of applications.