Difference between revisions of "Team:Vilnius-Lithuania/Description"

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        <h1>Description</h1>
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    <p><strong>Fig. 1</strong> Schematic overview of the SynDrop</p>
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    <p></p>
        <p></p>
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    <h2><var>“What I cannot create, I do not understand”</var></h2>
        <h2>What is SynORI?</h2>
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    <h6>R. Feynmann, February 1988</h6>
        <p>SynORI stands for synthetic origin of replication. It is a framework designed to make working with single
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    <p></p>
            and multi-plasmid systems precise, easy and on top of that - more functional.</p>
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    <p><h2>Brief overview of the SynDrop - Synthetic Droplets for Membrane Protein Research.</h2></p>
        <p>The SynORI framework enables scientists to build a multi-plasmid system in a standardized manner by:</p>
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    <p></p>
        <ol>
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    <p>
            <li>Selecting the number of plasmid groups</li>
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            SynDrop started from the idea of working towards developing a minimal synthetic cell. However it was soon realized that synthetic life is not something that will be made in one go - it will be the culmination of all the small, separate systems that will come together and work in unison. As such, it was understood that these systems need to be independently functional and well described first, before more complex systems are built on top of them. One of the most fundamental differences between life and synthetic systems is the responsivity and communication with the surrounding environment. This function in living cells is mostly performed by membrane proteins. We quickly realized that in order to make a significant impact on synthetic life development, membrane proteins are that understudied field that holds great potential for future applications in synthetic biology. Fig. 1 beautifully summarizes the workflow, complexity and at the same time minimalism of SynDrop. We utilized the emerging technology of microfluidics to synthesize cell-sized liposomes and provide them with the minimal set of all the necessary tools and machineries for the successful synthesis of membrane proteins. These fully equipped liposomes form the core of the SynDrop. Within them are encapsulated purified BAM complex proteins and the chaperone SurA which facilitate beta-barrel bearing protein assembly. Liposomes also contain genetically engineered membrane-associating ribosomes which increase the yields of target protein expression. SynDrop liposomes contain an <var>in vitro</var> transcription-translation system and custom DNA. Their inner aqueous environment is suitable for molecular reactions to occur. Finally, SynDrop provides a novel platform for protein display, whether they were antibodies, single chain fragments, globular proteins, or peptides. It is a huge step forward in membrane protein research and perhaps another resolved puzzle towards the creation of synthetic cell.
            <li>Choosing the copy number of each group</li>
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            <li>Picking the type of copy number control (specific to one group or regulating all of them at once).</li>
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        </ol>
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    </p>
        </p>
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    <p></p>
 
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    <h1>Background</h1>
        <p></p>
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    <p></p>
        <p>The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics
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            (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low
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            copy number plasmid groups are not lost during the division.
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        </p>
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        <p></p>
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        <div class="img-cont">
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            <img src="https://static.igem.org/mediawiki/parts/8/84/Collect.png" alt="img">
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            <div class="img-label">
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            </div>
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        </div>
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        <h2>Applications</h2>
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        <p>
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            <h5>Everyday lab work</h5>
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            <p>
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                A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your
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                research to a particular plasmid copy number just because there are not enough right replicons to
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                choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that
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                suits your needs.</li>
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            </p>
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            <h5>Biological computing</h5>
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            <p>
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                The ability to choose a wide range of copy number options and their control types will make the
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                synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number
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                regulation is equivalent to adding a global parameter to a computer system. It enables the coordination
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                of multiple gene group expression.
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            </p>
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            <h5>Smart assembly of large protein complexes</h5>
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            <p>
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                The co-expression of multi-subunit complexes using different replicons brings incoherency to an already
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                chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses
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                the same type of control, and in addition can act in a group-specific manner.</p>
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            <h5>Metabolic engineering</h5>
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            <p>
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                A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the
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                levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in
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                transcriptional control and synthetic ribosome binding sites in translational control are already
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                widely used to maintain expression levels. In addition to current approaches, our framework allows a
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                simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the
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                search for perfect conditions a lot easier.
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            </p>
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        </p>
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        <p>
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        </p>
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        <table style="width:100%">
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<thead>
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<td align='center'>Species sign in ODE system</td>
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<td align='center'>Species</td>
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<td align='center'>Initial concentration (M)</td>
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</thead>
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<tbody>
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<tr>
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<td align='center'>A</td>
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<td align='center'>pDNA+RNA I+RNAII early</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>B</td>
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<td align='center'>pDNA+RNA II short</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>RNAI</td>
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<td align='center'>RNA I</td>
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<td align='center'>1E-6</td>
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</tr>
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<tr>
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<td align='center'>D</td>
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<td align='center'>pDNA+RNA II long</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>E</td>
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<td align='center'>pDNA+RNAII primer</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>F</td>
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<td align='center'>RNA II long</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>G</td>
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<td align='center'>pDNA</td>
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<td align='center'>4E-8*</td>
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</tr>
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<tr>
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<td align='center'>H</td>
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<td align='center'>pDNA+RNA II+RNA I late</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>RNA II</td>
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<td align='center'>RNA II</td>
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<td align='center'>0</td>
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</tr>
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<tr>
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<td align='center'>J</td>
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<td align='center'>RNAI+RNAII</td>
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<td align='center'>0</td>
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</tr>
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</tbody>
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</table>
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<a href="http://someurl.com">CLICK ME</a>
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<object data="https://static.igem.org/mediawiki/2017/6/6d/T--Vilnius-Lithuania--labnote.pdf" type="application/pdf" width="100%" height="700px" internalinstanceid="5" title="">
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  <p>It appears you don't have a PDF plugin for this browser, you can <a href="https://static.igem.org/mediawiki/2017/9/99/T--Vilnius-Lithuania--ExhibitionProtocol2.pdf">click here to
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  download the PDF file.</a></p></object>
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<video width="100%" height="240" controls>
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<source src="https://static.igem.org/mediawiki/2018/f/fe/IGEM_Human_Practices.mp4" type="video/mp4">
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</video>
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Revision as of 02:12, 18 October 2018

Description

Describe the Impossible

Cell-free systems are becoming an increasingly popular in vitro tool to study biological processes as it is accompanied by less intrinsic and extrinsic noise. Relying on fundamental concepts of synthetic biology, we apply a bottom-up forward engineering approach to create a novel cell-free system for unorthodox protein-evolution. The core of this system is cell-sized liposomes that serve as excellent artificial membrane models. By encapsulating genetic material and full in vitro protein transcription and translation systems within the liposomes, we create reliable and incredibly efficient nanofactories for the production of target proteins. Even though there are many alternative proteins that can be synthesized, our main focus is directed towards membrane proteins, which occupy approximately one third of living-cells’ genomes. Considering their significance, membrane proteins are spectacularly understudied since synthesis and thus characterization of them remain prevailing obstacles to this day. We aim to utilize liposomes as nanofactories for directed evolution of membrane proteins. Furthermore, by means of directed membrane protein-evolution, a universal exposition system will be designed in order to display any protein of interest on the surface of the liposome. This way, a system is built where a phenotype of a particular protein is expressed on the outside while containing its genotype within the liposome. To prove the concept, small antibody fragments will be displayed to create a single-chain variable fragment (scFv) library for rapid screening of any designated target.

invert