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

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<h1>Membrane Integration</h1>
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<h1>Collaboration</h1>
 
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<h2>New proteins for the peroxisomal membrane</h2>
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<h2>Collaborating with Team Aachen</h2>
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<h3>Membrane Integration</h3>
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<p>Many reactions rely on optimal conditions like pH and [...]. 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-dependent and an ER-dependent one [1,2].</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 [3]. 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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<p>As a proof of concept, we will incorporate three proteins through three different approaches into the peroxisomal membrane: (i) mRuby2-PEX26 as a proof for the Pex19-dependent mechanism, (ii) Pex3-mRuby2 itself to showcase to ER-dependent mechanism and (iii) Bacteriorhodopsin, a unidirectional proton pump, fused to the N-terminal anchor of Pex3. </p>
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<h4>Pex19-dependent Mechanism</h4>
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<p>The exact mechanisms of mPTS binding, Pex3/Pex19 disassembly, mPTS-PMP binding, and release from the Pex3/Pex19 mediated mPTS-PMP docking to the full integration into the membrane are yet unknown [4]. However, general principles of the integration of a new peroxisomal membrane protein (PMP) through Pex19 and Pex3 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 Pex3p, 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. [3]</p>
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<h4>Experimental Work/Design</h4>
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<h4>Research and references</h4>
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Revision as of 18:23, 30 October 2017

Collaboration

Collaborating with Team Aachen

Like in all other fields of modern science, collaboration between different working groups is a beneficial way to combine experience and knowledge in order to achieve proper project results. For that reason, iGEM teams from all over the world collaborate to help each other with complex and challenging tasks on their way of accomplishing their specific project aims. In this year, our team joined forces with Aachen in order to improve both of our projects.

Since the principle purpose of our project was to design and create an artificial compartment, orthogonal peroxisomal protein import is one major part of achieving our objective. For that reason, we needed to deactivate natural protein import facilitated by the two receptor proteins Pex5 and Pex7. Initially we already possessed yeast strains which are deficient for Pex5 or Pex7, but we additionally planned to create a yeast cell line which is unable to express both import proteins, resulting in peroxisomes lacking luminal proteins which are called ‘ghosts’. Subsequently, we will integrate our own protein translocation system, allowing us to completely control the protein content of our artificial compartment. We consulted iGEM Aachen which already had lots of experience and knowledge regarding gene knockouts in Saccharomyces cerevisiae. The double knockout was performed successfully by team Aachen, allowing us to get rid of the natural peroxisomal protein import machinery. Conclusively, through this collaboration with team Aachen we were able to improve our final results significantly.

Thanks to the double knockout for the import proteins Pex5 and Pex7 we obtained completely empty ghosts. Thus, verification of the function of our orthogonal peroxisomal protein import machinery was feasible. We therefore accomplished to transfer our artificial Pex5 ‘artico’-receptor including our modified binding pocket region recognizing our modelled PTS1 peptide (PTS1*). When fused to the fluorescent marker protein mTurquoise, our artificial Pex5 facilitates peroxisomal import of the mTurquoise-PTS1* fusion protein indicated by 3D-Sim microscopy.

In return to the help we received from team Aachen we were able to provide them with different plasmids to significantly improve their final project. In the beginning of our collaboration with team Aachen we heard about their problems with building optimized yeast plasmids. For that reason, we introduced them to the yeast toolbox we used which enables the possibility to simplify the process of engineering yeast by creating completely designed plasmids facilitating optimal gene expression and translation for particular purposes in S. cerevisiae. With our golden gate cloning assembly, we designed four different plasmids enabling testing of different promoter strengths for a chloride channel from Arabidopsis thaliana (ATCLCc). We successfully built three yeast optimized plasmids containing the ATCLCc gene with different promotors (Gal1, Sac6, RPL18B) and a fourth plasmid for genome integration containing URA 3’ and 5’ homologies. Correctness of each plasmid was verified by performing restriction digests and sanger sequencing, respectively.

Once we finished the cloning and verification of each plasmid we gave them to team Aachen, allowing them to improve their project results significantly by providing the availability of another vacuolar anion transporter. Thanks to our effort, iGEM Aachen was able to utilize this chloride channel in their yeast based system used for water preparation properly.

Optogenetics seminar

Optogenetics offers high spatiotemporal control of well defined events within living cells and therefore qualifies for a broad range of synthetic biological applications. Dusseldorfs last year’s team successfully used optogenetics to induce apoptosis in cancer cells and given that experience, we decided to arrange an optogenetics seminar to share our knowledge with other teams. With that, we hope to reach as many iGEM teams as possible in order to provide essentials in performance of optogenetic experiments. Additionally, we wanted to extend our iGEM teams “toolbox” with one more tool of control.
Our offer found international approval by the teams of Darmstadt, Heidelberg, Paris and Taiwan. Since the teams of Paris and Taiwan could not attend the seminar in person, they joined us via skype.
The seminar started with a marvelous introduction given by Patrick Fischbach. Afterwards, we took advantage of the situation and presented each team’s project, followed by breathtaking discussions facing the difficulties and odds of optogenetic in their project. Introducing the practical handling with optogenetics, Tim Blomeier, who is an advisor of our team, gave a talk about working with optogenetics. With that we imparted all necessary aspects one has to consider when working with optogenetics. Nevertheless nothing is more helpful than practical experience - to attain that, we showed the attendants our laboratory, as well as our hardware and the dark room for optogenetic experiments. At the end of the day we had a nice barbecue. This comfortable situation offers the possibility for lot of interesting discussions regarding optogenetic, our projects and iGEM itself.